Complex Coacervate for Controlled Release and Related Methods

ABSTRACT

Provided herein are coacervate compositions including cytokines, and methods of making and using the same. The coacervate can be formed by the mixing of an active agent, such as a drug or protein with the polyanion, such as heparin or heparan sulfate, and a custom-made polycation (e.g., PEAD or PELD). The coacervates can be used in the treatment of diseases and disorders where targeted treatment is desired, for example in treatment of cancers.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/060,075, filed Jun. 7, 2018, which is the United Statesnational phase of International Application No. PCT/US2016/066640 filedDec. 14, 2016, and claims the benefit of U.S. Provisional PatentApplication No. 62/266,896, filed Dec. 14, 2015, each of which isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under Grant NosDMR1005766 and IIP1444774, awarded by the National Science Foundation.The government has certain rights in the invention.

SEQUENCE LISTING STATEMENT

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 6527_1803711_ST25.txt. The size ofthe text file is 1828 bytes, and the text file was created on Jun. 7,2018.

BACKGROUND OF THE INVENTION

Provided herein are compositions useful for drug delivery, for examplefor delivery of cytokines, and methods of use of those compositions, forexample for treatment of coronary heart disease and cancer.

Cytokines are produced by a broad range of cell types and serve assoluble modulators of immune function in vitro and in vivo. In thecancer setting, interleukins (IL), such as IL-2 and IL-12, andinterferons (IFN), such as IFN-α and IFN-γ, are capable of promotingprotective anti-tumor immunity in patients with solid forms of cancer.However, systemic delivery of high-doses of these agents over prolongedperiods of time has resulted in severe toxicities, and even patientdeaths.

Coronary heart disease (CHD) affects 15.4 million Americans and is themost common type of heart disease. CHD alone accounts for 385,000 deathsand costs an estimated total of $108.9 billion annually (direct andindirect) in the United States. CHD caused by pathological blockage ofthe coronary circulation may lead to prolonged ischemia which in turnresults in permanent cardiomyopathy and/or myocardial infarction (MI).MI causes death of cardiac myocytes and triggers local inflammatoryresponses and the compensatory scar formation, leading to pathologicalremodeling and ultimately heart failure (HF). Recent experimentaltherapies for cardiac repair primarily focus on revascularization andregeneration of impaired myocardium. However, to break the vicious cycleof MI-to-HF, not only is revascularization of the ischemic tissuedesirable, but also modulation or the over-activated and prolongedinflammation following myocardial injury.

A safe and effective method of delivery of cytokines is needed fortreatment of patients, for example with coronary heart disease andcancer.

SUMMARY

Systemic toxicities of cytokines can be avoided by the directed deliveryof these cytokines in a local manner, i.e. into the treatment site.Provided herein is a controlled delivery coacervate made of acombination of a polyanion, such as heparin or heparan sulfate and asynthetic polycationic copolymer. The coacervate is formed by the mixingof an active agent, such as a drug or protein with the polyanion, suchas heparin or heparan sulfate, and a custom-made polycation (e.g., PEADor PELD). Complex coacervates are formed by mixing oppositely chargedpolyelectrolytes resulting in spherical droplets of organic moleculesheld together noncovalently and apart from the surrounding liquid. Thecoacervate system provides a higher level of control over the release ofdrugs from a delivery system. Embedding a drug in coacervatecompositions leads to the release of the drug over days to months. Slowrelease of drugs also is desirable when timing of delivery impactstreatment—for example, to prevent release of a large bolus of cytokinesto a patient, and to normalize delivery of cytokines over a much longertime period than has been possible using conventional delivery vehiclessuch as saline.

Provided is a composition comprising a complex or coacervate of apolycationic polymer, a polyanionic polymer, such as heparin or heparansulfate and a cytokine selected from an interferon and/or aninterleukin. In one aspect, the polycationic polymer is PEAD or PELD, orpolymer composition comprising at least one moiety selected from thefollowing:

-   -   (a)        [—OC(O)—CH(NHY)—CH₂—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]_(n),    -   (b)        [—OC(O)—CH₂—CH(NHY)—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]n,    -   (c)        [—OC(O)—CH(NHY)—CH₂—CH₂—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]_(n),        and/or    -   (d)        [—OC(O)—CH₂—CH₂—CH(NHY)—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]n,        wherein Y is —C(O)—CH(NH₃ ⁺)—(CH₂)₃—NH—C(NH₂)₂ ⁺ or —C(O)—CH(NH₃        ⁺)—(CH₂)⁴—(NH₃)⁺, and R1 and R2 are the same or different and        are independently selected from the group consisting of        hydrogen, a carboxy-containing group, a C₁₋₆ alkyl group, an        amine-containing group, a quaternary ammonium containing group,        and a peptide. In one aspect, the cytokine is immunomodulatory.        In another aspect, the cytokine is IL-12. In another, the        cytokine is IL-10, and the composition optionally further        comprises an angiogenic growth factor such as FGF2. In another        aspect, the composition is embedded in a hydrogel.

Also provided are methods of treatment of coronary heart disease, suchas a myocardial infarction comprising administering to a patient in needthereof an effective amount of the composition described above, andherein, e.g., including effective amounts of IL-10 and FGF2. Further, amethod of treatment of a cancer is provided, comprising administering toa patient in need thereof an effective amount of the compositiondescribed above and herein, e.g., including an effective amount of anIL-12.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F. Characterization of FGF2/IL-10 coacervate. (FIG. 1A)Schematic illustration of putative FGF2/IL-10 coacervate structure (PEADin blue in original, heparin in green in original, FGF2 in yellow inoriginal, and IL-10 in orange in original). Please note that FGF2 andIL-10 molecules bound to heparin are circled with green dashed lineswhile FGF2 and IL-10 molecules physically entrapped within thecoacervate structure (no affinity-based binding) are circled with dashedline. (FIG. 1B) Analysis of coacervate droplet size with polydispersityindex (PDI). (FIG. 1C) The release profile of FGF2/IL-10 coacervate invitro for 3 weeks. Equal amount of FGF2 and IL-10 were first combinedand then mixed with heparin, followed by the addition of PEAD to formcoacervate, with a mass ratio of PEAD:heparin:FGF2:IL-10=500:100:1:1.Supernatants were collected on Day 0, 0.5, 1, 4, 7, 10, 14, and 21 andthen replenished with PBS containing fresh 0.5 U/mL heparinase II inorder to simulate the release of cargo proteins in vivo (N=4 per timepoint). The amount of released FGF2 and IL-10 was measured by ELISA.Data are presented as percent cumulative release (normalized to theoriginal load). Error bars indicate means±SD. (FIG. 1D) Incorporation offluorescently labeled FGF2 (green in original) and IL-10 (red inoriginal) into coacervate droplets (scale bar=25 μm) (FIG. 1E)High-magnification enlargement of the coacervate droplet in the dottedarea in (FIG. 1D) showing fluorescently labeled FGF-2 and IL-10molecules evenly distributed within the droplet (scale bar=5 μm). (FIG.1F) Triply labeled heparin (red in original), FGF2 (green in original)and IL-10 (blue in original) showing nearly homogeneous structure ofFGF-2/IL-10 coacervate (scale bar=10 μm). Please note that due to thelimitation of imaging resolution, larger coacervate droplets were chosenfor high-magnification imaging.

FIG. 2. The spontaneous degradation of free IL-10 in vitro. Thedegradation profile of free IL-10 (1 μg/mL) in PBS containing 0.5 U/mLheparinase II was measured for 7 days. Supernatants were collected onDay 0, 1, 2, 3, 5, and 7 and stored at −80° C. for simultaneous ELISAanalysis (N=4 per time point). A spontaneous loss of free IL-10 wasdetected at an average rate of 1.59% per day.

FIG. 3. Bioactivity of FGF-2/IL-10 coacervate on cardiac stromal cellproliferation in vitro. Bioactivity of FGF2/IL-10 coacervate on cardiacstromal cell proliferation was tested in a non-contact transwell systemfor 72 hours with human umbilical vein endothelial cells (HUVECs), humancardiac fibroblasts (hCFs), and human heart pericytes (hHPs), and mousecardiac fibroblasts (mCFs) cultured at bottom wells and treatmentsolutions loaded into suspended transwells. (A) Under the simpleserum-deprived condition, Free-F/I-500/100, Coa-F-500, Coa-F/I-500/100and Coa-F/I-500/500 had significantly higher HUVEC proliferation thanthe no-treatment control and Free-F/I-500/500. Coa-F/I-500/500 hadsignificantly lower mCF proliferation than the no-treatment control,Free-F/I-500/100, and Free-F/I-500/500 while Coa-F/I-500/100 hadsignificantly lower mCF proliferation than Free-F/I-500/100 andFree-F/I-500/500. Free-F/I-500/100, Free-F/I-500/500, andCoa-F/I-500/500 marginally promoted hHP proliferation (all p>0.05). (B)Under the serum-deprived condition with inflammatory stress (10 ng/ml ofTNF-α for HUVECs and 100 ng/ml of TNF-α for all other cell types),Coa-F-500, Coa-F/I-500/100, and Coa-F/I-500/500 had significantly higherHUVEC proliferation than the no-treatment control. Coa-F-500,Coa-F/I-500/100, and Coa-F/I-500/500 had significantly lower hCFproliferation than Free-F/I-500/100. Additionally, Coa-F/I-500/500 hadsignificantly lower mCF proliferation than the no-treatment control,Free-F/I-500/100, and Free-F/I-500/500 while Coa-F-500 andCoa-F/I-500/100 had significantly lower mCF proliferation than theno-treatment control and Free-F/I-500/100. Error bars indicate means±SD.Statistical differences between groups were analyzed by one-way ANOVAwith Bonferroni post-hoc analysis. (*p<0.05, †p<0.01, § p<0.005,#p<0.001 in all graphs) (From left-to-right in each group in Figure;No-Tc Ctrl: basal medium; Vehicle: empty [PEAD:heparin]coacervate;Free-F/I-500/100: naked mixture of 500 ng FGF2 and 100 ng IL-10;Free-F/I-500/500: naked mixture of 500 ng FGF2 and 500 ng IL-10;Coa-F-500: coacervate loaded with 500 ng FGF2; Coa-F/I-500/100:coacervate loaded with 500 ng FGF2 and 100 ng IL-10; Coa-F/I-500/500:coacervate loaded with 500 ng FGF2 and 500 ng IL-10)

FIGS. 4A-4E. Intramyocardial injection of FGF2/IL-10 coacervate improveslong-term cardiac contractility and ameliorates adverse remodeling.Echocardiographic analyses (N=8 per group except Free-F/I-500/500, N=7)were repeatedly performed at 5 days, 2 and 6 weeks post-surgery.Statistical differences in overall treatment effect were analyzed bytwo-way repeated ANOVA with Bonferroni multiple comparison test. Theresults revealed substantial improvement in LV contractility followingintramyocardial injection of either Coa-F/I-500/100 or Coa-F/I-500/500,as indicated by the higher (FIG. 4A) fractional shortening (FS), (FIG.4B) fractional area change (FAC), and (FIG. 4C) ejection fraction (EF)(*p<0.05, †p<0.01, § p<0.005, #p<0.001 in all graphs; FS:Coa-F/I-500/100 and Coa-F/I-500/500 vs. Saline, Free-F/I-500/500, andCoa-F-500; FAC and EF: Coa-F/I-500/500 vs. all groups, Coa-F-500 andCoa-F/I-500/100 vs. Saline and Free-F/I-500/500, Free-F/I-500/500 vs.Saline). Significant reductions of (FIG. 4D) end-diastolic area (EDA)and (FIG. 4E) end-systolic area (ESA) of LV were observed in heartstreated with Coa-F/I-500/100 or Coa-F/I-500/500 (*p<0.05, †p<0.01, §p<0.005, #p<0.001 in all graphs; EDA: Coa-F/I-500/500 vs. Saline,Free-F/I-500/500, and Coa-F-500; Coa-F/I-500/100 vs. Saline andFree-F/I-500/500; ESA: Coa-F/I-500/500 vs. Saline, Free-F/I-500/500, andCoa-F-500; Coa-F-500 and Coa-F/I-500/100 vs. Saline andFree-F/I-500/500). Error bars indicate means±SD. Please note that timepoints on the X-axis (time) in all graphs are not scaled to actualexperimental duration.

FIGS. 5A and 5B. FGF-2/IL-10 coacervate amends elasticity of infarctedmyocardium. (FIG. 5A) Representative axial strain maps laid over B-modeimages (4×6 mm) showing the axial strain distribution of the normal(left panel) and untreated MI control (right panel, mid-infarct level)left ventricles respectively. For normalization purpose, the infarctarea was designated as B, and the non-infarct area was designated as A.(FIG. 5B) Normalized strain was obtained by dividing spatially averagedaxial strain of B by that of A (B/A). Coa-F-500, Coa-F/I-500/100, andCoa-F/I-500/500 showed markedly greater normalized strains than thesaline control and Free-F/I-500/500. Free-F/I-500/500 also showednotably higher normalized strains than the saline control (#p<0.001,†p<0.01; N=3 per group). Error bars indicate means±SD. Statisticaldifferences between groups were analyzed by one-way ANOVA withBonferroni post-hoc analysis.

FIG. 6. B-mode images of normal and untreated MI control hearts withoutstrain maps and ROIs.

FIGS. 7A and 7B. FGF-2/IL-10 coacervate increases long-term endothelialcell density. Endothelial cell (EC) density at the infarct andperi-infarct border zone was revealed by immunohistochemical detectionof CD31+ ECs at 6 weeks post-infarction. (FIG. 7A) Representative imagesof CD31+ ECs (red in original) and αSMA+ cells (green in original)within the infarct and peri-infarct areas at the mid-infarct level, withdotted areas enlarged. Please note that vascular smooth muscle cells(VSMC) were defined as perivascular/peri-CD31 αSMA+ cells. Nuclei werestained with DAPI in blue (in original). (scale bars=100 μm) (FIG. 7B,left) Quantitative analyses of CD31+ EC density within the infarct arearevealed that Coa-F/I-500/500 had significantly higher EC density thanthe saline control and Free-F/I-500/500 while Coa-F/I-500/100 hadsignificantly higher EC density than the saline control (*p<0.05,†p<0.01; N=4 per group). (FIG. 7B, right) Within the peri-infarct area,Coa-F/I-500/500 had significantly higher EC density than the salinecontrol, Free-F/I-500/500, and Coa-F-500 while Coa-F/I-500/100 hadsignificantly higher EC density than the saline control andFree-F/I-500/500 (#p<0.001, *p<0.05; N=4 per group). Error bars indicatemeans±SD. Statistical differences between groups were analyzed byone-way ANOVA with Bonferroni post-hoc analysis.

FIG. 8. FGF-2/IL-10 coacervate enhances long-term vascular stromal celldensity. Vascular stromal cell density at the infarct and peri-infarctborder zone was revealed by immunohistochemical detection of vascularsmooth muscle cells (VSMC, defined as perivascular/peri-CD31 αSMA+cells; representative images shown in FIG. 7(A)) at 6 weekspost-infarction. (left) Quantitative analyses of perivascular αSMA+VSMCdensity within the infarct area revealed that Coa-F/I-500/500 hadsignificantly higher VSMC density than the saline control,Free-F/I-500/500, and Coa-F-500 (*p<0.05; N=4 per group). (right)Coa-F/I-500/100 and Coa-F/I-500/500 had marginally higher VSMC densitythan the saline control, Free-F/I-500/500, and Coa-F-500 within theperi-infarct area (all p>0.05; N=4 per group). Error bars indicatemeans±SD. Statistical differences between groups were analyzed byone-way ANOVA with Bonferroni post-hoc analysis.

FIG. 9. FGF-2/IL-10 coacervate reduces myocardial fibrosis. Masson'strichrome histological staining was employed to reveal left ventricular(LV) myocardial fibrosis at 6 weeks post-infarction. (A) Representativeimages of myocardial fibrosis at the mid-infarct level (transversesections of hearts). Collagen deposition (fibrosis/scar) was stained inblue/purple while cardiac muscle was stained in red (scale bars=1 mm).(B) Quantification of the LV fibrotic area fraction. Coa-F/I-500/500exhibited significantly reduced LV fibrotic area fraction (#p<0.001, vs.saline; N=4 per group). Healthy heart (Normal) served as a negativecontrol. (C) Measurement of LV wall thickness at the center of theinfarct. Coa-F/I-500/500 had significantly thicker infarct wall than allother groups (*p<0.05, vs. all groups; N=4 per group). Error barsindicate means±SD. Statistical differences between groups were analyzedby one-way ANOVA with Bonferroni post-hoc analysis.

FIG. 10. FGF-2/IL-10 coacervate inhibits chronic phagocytic cellinfiltration The effect of FGF-2/IL-10 coacervate on chronicinflammatory responses was evaluated by the number of focallyinfiltrating CD68+ phagocytic cells within the infarct region at 6 weekspost-infarction. When compared with the saline control, Coa-F-500,Coa-F/I-500/100, and Coa-F/I-500/500 had significantly reduced numbersof infiltrated CD68+ phagocytic cells within the infarct area. Errorbars indicate means±SD. Statistical differences between groups wereanalyzed by one-way ANOVA with Bonferroni post-hoc analysis. (*p<0.05,†p<0.01, vs. saline; N=4 per group).

FIG. 11. Estimation of the duration of coacervate treatment in vivo.Multi-photon excitation (MPE) imaging was employed to detectintramyocardially injected free (Free-Rho), heparin-bound (Hep-Rho), orcoacervate-bound rhodamine (Coa-Rho). Collagen fibers (blue in original)were revealed by second harmonic generation (SHG) signals.Quantification of the fluorescence volume of Free-Rho, Hep-Rho, andCoa-Rho at 5, 14, and 28 days post-injection. Error bars indicatemeans±SD. Statistical differences between groups were analyzed byone-way ANOVA with Bonferroni post-hoc analysis. (*p<0.05, vs. Free-Rhoand Hep-Rho; N=3 per group).

FIG. 12 depicts postulated mechanisms of FGF-2/IL-10 coacervate-mediatedcardiac repair and functional recovery.

FIG. 13 is a graph depicting survival proportions as described inExample 5.

FIG. 14 provides graphs showing a summary of results for Example 5(IL-2).

FIGS. 15A-15C are graphs showing the results for individual mice forExample 5 (IL-2).

FIG. 16 provides graphs showing a summary of results for Example 5(IL-12).

FIGS. 17A-17C are graphs showing the results for individual mice forExample 5 (IL-12).

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values. For definitions provided herein, those definitionsrefer to word forms, cognates and grammatical variants of those words orphrases.

A composition is provided herein to control the delivery rate ofcytokines, for example, interleukins (IL), such as IL-2 and IL-12 (e.g.,IL-12 p70), and interferons (IFN), such as IFN-γ. The controlleddelivery system comprises a recently developed heparin-based coacervate.Complex coacervates are formed by mixing oppositely chargedpolyelectrolytes resulting in spherical droplets of organic moleculesheld together noncovalently and apart from the surrounding liquid andhave shown potential in sustained protein delivery. One application ofthe coacervate is to control the release of cytokines and interferons.Methods of making and using the composition also are provided.

As used herein, the term “polymer composition” is a compositioncomprising one or more polymers. As a class, “polymers” includeshomopolymers, heteropolymers, co-polymers, block polymers, blockco-polymers and can be both natural and synthetic. Homopolymers containone type of building block, or monomer, whereas co-polymers contain morethan one type of monomer.

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, are meant to be open ended. The terms “a” and “an”are intended to refer to one or more.

As used herein, the term “patient” or “subject” refers to members of theanimal kingdom including but not limited to human beings.

A “coacervate” refers to herein as a reversible aggregation ofcompositions in a liquid, for example, as described herein, for example,resulting from the aggregation of oppositely-charged polyioniccompositions. Exemplary coacervates are illustrated in the examplesbelow with the aggregation of the polycation, polyanion, and activeagent(s), as described herein, for example with the aggregation of PEAD,Heparin, and IL-12, or IL-10 combined with FGF2. A “complex” is anon-covalent aggregation of two or more compositions.

The term “alkyl” refers to both branched and straight-chain saturatedaliphatic hydrocarbon groups. These groups can have a stated number ofcarbon atoms, expressed as C_(x-y), where x and y typically areintegers. For example, C₅₋₁₀, includes C₅, C₆, C₇, C₈, C₉, and C₁₀.Alkyl groups include, without limitation: methyl, ethyl, propyl,isopropyl, n-, s- and t-butyl, n- and s-pentyl, hexyl, heptyl, octyl,etc. Alkenes comprise one or more double bonds and alkynes comprise oneor more triple bonds. These groups include groups that have two or morepoints of attachment (e.g., alkylene). Cycloalkyl groups are saturatedring groups, such as cyclopropyl, cyclobutyl, or cyclopentyl. As usedherein, “halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

A polymer “comprises” or is “derived from” a stated monomer if thatmonomer is incorporated into the polymer. Thus, the incorporated monomerthat the polymer comprises is not the same as the monomer prior toincorporation into a polymer, in that at the very least, certainterminal groups are incorporated into the polymer backbone. A polymer issaid to comprise a specific type of linkage, such as an ester, orurethane linkage, if that linkage is present in the polymer.

Certain polymers described herein, such as heparin and PEAD, are said tobe bioerodible or biodegradable. By that, it is meant that the polymer,once implanted and placed in contact with bodily fluids and tissues, orsubjected to other environmental conditions, such as composting, willdegrade either partially or completely through chemical reactions,typically and often preferably over a time period of hours, days, weeksor months. Non-limiting examples of such chemical reactions includeacid/base reactions, hydrolysis reactions, and enzyme catalyzed bondscission. Certain polymers described herein contain labile esterlinkages. The polymer or polymers may be selected so that it degradesover a time period. Non-limiting examples of useful in situ degradationrates include between 12 hours and 5 years, and increments of hours,days, weeks, months or years therebetween.

A drug delivery composition is provided, comprising, a coacervate of apolycationic polymer, a polyanionic polymer, and an active agent. Incertain aspects, the polycationic polymer described herein comprises thestructure (that is, comprises the moiety: [—OC(O)—B′—CH(OR1)-B-]_(n) or—[OC(O)—B—C(O)O—CH₂—CH(O—R1)-CH₂—B′—CH₂—CH(O—R2)-CH₂-]_(n), in which Band B′ are the same or different and are organic groups, or B′ is notpresent, including, but not limited to: alkyl, ether, tertiary amine,ester, amide, or alcohol, and can be linear, branched or cyclic,saturated or unsaturated, aliphatic or aromatic, and optionally compriseone or more protected active groups, such as, without limitation,protected amines and acids, and R1 and R2 are the same or different andare hydrogen or a functional group (e.g., as described herein). As seenbelow, the composition exhibits low polydispersity, with apolydispersity index of less than 3.0, and in many cases less than 2.0.These compositions are described in U.S. Pat. No. 9,023,972, which isincorporated by reference in its entirety.

In one aspect, the polycationic polymer is a polymer compositioncomprising at least one moiety selected from the following in which Band B′ are residues of aspartic acid or glutamic acid, which areoptionally further derivatized with an amine-containing group, forexample, the amines of the aspartic acid or glutamic acid are furtherderivatized with lysine or arginine:

-   -   (a)        [—OC(O)—CH(NHY)—CH₂—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]_(n),    -   (b)        [—OC(O)—CH₂—CH(NHY)—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]_(n),    -   (c)        [—OC(O)—CH(NHY)—CH₂—CH₂—C(O)O—CH2-CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]_(n),        and/or    -   (d)        [—OC(O)—CH₂—CH₂—CH(NHY)—C(O)O—CH2-CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]_(n),        wherein Y is —C(O)—CH(NH₃ ⁺)—(CH₂)₃—NH—C(NH₂)₂ ⁺ or —C(O)—CH(NH₃        ⁺)—(CH₂)₄—(NH₃)⁺, and R1 and R2 are the same or different and        are independently selected from the group consisting of        hydrogen, a carboxy-containing group, a C₁₋₆ alkyl group, an        amine-containing group, a quaternary ammonium containing group,        and a peptide.

The polymers described herein can be functionalized, e.g., at B, B′, R1and R2, meaning they comprise one or more groups with an activity, suchas a biological activity. For example and without limitation, as shownherein, the polymer may be functionalized with an acetylcholine-likegroup or moiety, a cross-linking agent (cross-linking agents contain atleast two reactive groups that are reactive towards numerous groups,including sulfhydryls and amines, and create chemical covalent bondsbetween two or more molecules, functional groups that can be targetedwith cross-linking agents include primary amines, carboxyls,sulfhydryls, carbohydrates and carboxylic acids. A large number of suchagents are available commercially from, e.g., Thermo fisher Scientific(Pierce) and Sigma.

Other functions that can be provided by or enhanced by addition offunctional groups include: increased hydrophobicity, for instance byfunctionalizing with a superhydrophobic moiety, such as aperfluoroalkane, a perfluoro(alkylsilane), and/or a siloxane; increasedhydrophilicity, for instance by functionalizing with polyethylene glycol(PEG); or antimicrobial, for instance, by functionalizing with aquaternary amine. The polymer can be functionalized with a tag, such asa fluorescent tag (e.g., FITC, a cyanine dye, etc.). The polymer can befunctionalized by linking to additional synthetic or natural polymers,including, without limitation: synthetic polymers, such as a polymerderived from an alpha-hydroxy acid, a polylactide, apoly(lactide-co-glycolide), a poly(L-lactide-co-caprolactone), apolyglycolic acid, a poly(dl-lactide-co-glycolide), apoly(1-lactide-co-dl-lactide), a polymer comprising a lactone monomer, apolycaprolactone, a polymer comprising carbonate linkages, apolycarbonate, a polyglyconate, a poly(glycolide-co-trimethylenecarbonate), a poly(glycolide-co-trimethylene carbonate-co-dioxanone), apolymer comprising urethane linkages, a polyurethane, a poly(esterurethane) urea, a poly(ester urethane) urea elastomer, a polymercomprising ester linkages, a polyalkanoate, a polyhydroxybutyrate, apolyhydroxyvalerate, a polydioxanone, a polygalactin, or naturalpolymers, such as chitosan, collagen, gelatin, elastin, alginate,cellulose, hyaluronic acid and other glycosaminoglycans.

The compositions may be functionalized with organic or inorganicmoieties to achieve desired physical attributes (e.g., hardness,elasticity, color, additional chemical reactivity, etc.), or desiredfunctionality. For example, the polymer composition may be derivatizedwith maleic acid or phosphate.

The composition is formed into a coacervate with active agents orpolyanionic or polycationic groups for sequestering active agents forcontrolled delivery in vivo. Drug products comprising the coacervatedescribed herein may be delivered to a patient by any suitable route ofdelivery (e.g. oral or parenteral), or as an implantable device for slowrelease of the active agent.

The functional groups may vary as indicated above. For example, incertain embodiments, R1 and R2 are the same or different and areindependently selected from the group consisting of hydrogen, acarboxy-containing group, a C₁₋₆ alkyl group, an amine-containing group,a quaternary ammonium containing group, and a peptide. In oneembodiment, one or more of B, B′, R1 and R2 are charged such that it ispossible to bind various water insoluble organic or inorganic compoundsto the polymer, such as magnetic inorganic compounds. As above, in oneembodiment, one or more of B, B′, R1 and R2 are positively charged. Inone embodiment, one or both of R1 and R2 are functionalized with aphosphate group. In another embodiment, the composition is attachednon-covalently to a calcium phosphate (including as a group, for exampleand without limitation: hydroxyapatite, apatite, tricalcium phosphate,octacalcium phosphate, calcium hydrogen phosphate, and calciumdihydrogen phosphate). In yet another embodiment, R1 and R2 areindependently one Ile-Lys-Val-Ala-Val (IKVAV) (SEQ ID NO: 1),Arg-Gly-Asp (RGD), Arg-Gly-Asp-Ser (RGDS) (SEQ ID NO: 2), Ala-Gly-Asp(AGD), Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) (SEQ ID NO: 3),Val-Ala-Pro-Gly-Val-Gly (VAPGVG) (SEQ ID NO: 4), APGVGV (SEQ ID NO: 5),PGVGVA (SEQ ID NO: 6), VAP, GVGVA (SEQ ID NO: 7), VAPG (SEQ ID NO: 8),VGVAPG (SEQ ID NO: 9), VGVA (SEQ ID NO: 10), VAPGV (SEQ ID NO: 11) andGVAPGV (SEQ ID NO: 12)).

In forming the composition (e.g., coacervate), the cationic polycationicpolymer is complexed with a polyanionic polymer, such as heparin orheparan sulfate, which is further complexed with an active agent, suchas a growth factor, small molecule, cytokine, drug, a biologic, aprotein or polypeptide, a chemoattractant, a binding reagent, anantibody or antibody fragment, a receptor or a receptor fragment, aligand, or an antigen and/or an epitope. Specific examples of activeagents include interleukins (IL), such as IL-2 and IL-12 (e.g., IL-12p70), and interferons (IFN), such as IFN-γ. In one aspect, thecomposition comprises a coacervate of a polycationic polymer comprisingone or more of moieties (a), (b), (c), and/or (d), as described above,and further comprising heparin or heparin sulfate complexed (that isnon-covalently bound) with the a first active agent, such as IL-2, IL-12(e.g., IL-12 p70), and/or IFN-γ, in any combination. The composition isformed, for example, by mixing in a suitable solvent, such as an aqueoussolution, such as water, saline (e.g. normal saline), or PBS, thepolyanionic, polycationic, and active agent constituents of thecomposition.

Additional active agents that may be incorporated into the coacervateinclude, without limitation, anti-inflammatories, such as, withoutlimitation, NSAIDs (non-steroidal anti-inflammatory drugs) such assalicylic acid, indomethacin, sodium indomethacin trihydrate,salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal,diclofenac, indoprofen sodium salicylamide, anti-inflammatory cytokines,and anti-inflammatory proteins or steroidal anti-inflammatory agents);antibiotics; anticlotting factors such as heparin, Pebac, enoxaparin,aspirin, hirudin, plavix, bivalirudin, prasugrel, idraparinux, warfarin,coumadin, clopidogrel, PPACK, GGACK, tissue plasminogen activator,urokinase, and streptokinase; growth factors. Other active agentsinclude, without limitation: (1) immunosuppressants; glucocorticoidssuch as hydrocortisone, betamethasone, dexamethasone, flumethasone,isoflupredone, methylprednisolone, prednisone, prednisolone, andtriamcinolone acetonide; (2) antiangiogenics such as fluorouracil,paclitaxel, doxorubicin, cisplatin, methotrexate, cyclophosphamide,etoposide, pegaptanib, lucentis, tryptophanyl-tRNA synthetase, retaane,CA4P, AdPEDF, VEGF-TRAP-EYE, AG-103958, Avastin, JSM6427, TG100801,ATG3, OT-551, endostatin, thalidomide, bevacizumab, neovastat; (3)anti-proliferatives such as sirolimus, paclitaxel, perillyl alcohol,farnesyl transferase inhibitors, FPTIII, L744, anti-proliferativefactor, Van 10/4, doxorubicin, 5-FU, Daunomycin, Mitomycin,dexamethasone, azathioprine, chlorambucil, cyclophosphamide,methotrexate, mofetil, vasoactive intestinal polypeptide, and PACAP; (4)antibodies; drugs acting on immunophilins, such as cyclosporine,zotarolimus, everolimus, tacrolimus and sirolimus (rapamycin),interferons, TNF binding proteins; (5) taxanes, such as paclitaxel anddocetaxel; statins, such as atorvastatin, lovastatin, simvastatin,pravastatin, fluvastatin and rosuvastatin; (6) nitric oxide donors orprecursors, such as, without limitation, Angeli's Salt, L-Arginine, FreeBase, nitrates, nitrites, Diethylamine NONOate, Diethylamine NONOate/AM,Glyco-SNAP-1, Glyco-SNAP-2, (.+−.)-S-Nitroso-N-acetylpenicillamine,S-Nitrosoglutathione, NOC-5, NOC-7, NOC-9, NOC-12, NOC-18, NOR-1, NOR-3,SIN-1, Hydrochloride, Sodium Nitroprusside, Dihydrate, Spermine NONOate,Streptozotocin; and (7) antibiotics, such as, without limitation:acyclovir, ofloxacin, ampicillin, amphotericin B, atovaquone,azithromycin, ciprofloxacin, clarithromycin, clindamycin, clofazimine,dapsone, diclazuril, doxycycline, erythromycin, ethambutol, fluconazole,fluoroquinolones, foscarnet, ganciclovir, gentamicin, itraconazole,isoniazid, ketoconazole, levofloxacin, lincomycin, miconazole, neomycin,norfloxacin, ofloxacin, paromomycin, penicillin, pentamidine, polymyxinB, pyrazinamide, pyrimethamine, rifabutin, rifampin, sparfloxacin,streptomycin, sulfadiazine, tetracycline, tobramycin, trifluorouridine,trimethoprim sulfate, Zn-pyrithione, and silver salts such as chloride,bromide, iodide and periodate.

Further examples of additional active agents include: basic fibroblastgrowth factor (bFGF or FGF-2), acidic fibroblast growth factor (aFGF),nerve growth factor (NGF), vascular endothelial growth factor (VEGF),hepatocyte growth factor (HGF), transforming growth factor-betapleiotrophin protein, midkine protein, platelet-derived growth factor(PDGF) and angiopoietin-1 (Ang-1). Active agents are included in thedelivery system described herein, and are administered in amountseffective to achieve a desired end-point, such as angiogenesis, tissuegrowth, inhibition of tissue growth, or any other desirable end-point.

According to one aspect, complex structures are provided that comprisethe coacervate described herein mixed with, distributed within, orotherwise combined with another composition, such as a hydrogel, apolymer, an inorganic substrate, a medical implant or device such as aprosthetic, a dosage form, a woven or non-woven mesh, etc. According toone aspect, the coacervate is combined with a hydrogel, for example byembedding the coacervate in a hydrogel. Such a structure is useful forproviding complex release profiles for active agents, for instance forpromoting specific tissue growth or as a timed-release dosage form. Insuch an aspect, one or more active agents are distributed by any methodin the coacervate and in the hydrogel so as to cause a defineddegradation and release pattern. One useful aspect would be to embed thecoacervate having a first active agent into a hydrogel, having a secondactive agent, to provide a complex release profile. In any aspect, theactive agent(s) can be any effective active agent(s), for example asdescribed above. As an example, factor A is embedded into a hydrogel,e.g. a fibrin gel, for early release and factor B is contained withinthe coacervate, for delayed release. For each indicated purpose it isnoted that appropriate relative amounts of the coacervate and hydrogelmay be used, as well as including effective amounts of the active agentsfor the intended purpose, respectively in the coacervate and hydrogel.Appropriate and effective amounts of each component can be determined inthe ordinary course by a person of skill in the art.

Examples of useful active agents and combinations of agents forincorporation into the described coacervate for treatment of cancersinclude: IL2, IL-12, and IFNγ and combinations thereof. Also describedherein is a method of treatment of myocardial infarction, using thecombination of IL-10 and fibroblast growth factor-2 (FGF-2) in thedescribed coacervate.

The coacervate composition is delivered in any manner useful fortreatment of a condition in a patient, such as for treatment ofcardiovascular disease or cancer, such as by enteral, parenteral, ortopical routes, for example and without limitation by: intravenous (IV),local injection, intramuscular, intracerebral, subcutaneous, orally,inhalation, topically, enema, intravaginal, intrauterine, ocular, orotic routes.

Suitable excipients or carriers are employed for delivery of thecoacervate composition, though the excipients are consistent withmaintenance of the coacervate complex. Suitable excipients arebroadly-known in the pharmaceutical arts, and include: solvents, such aswater, phosphate-buffered saline (PBS), saline; buffers; salts; acids;bases; rheology modifiers; chelating agents; colorants; flavorings;penetration enhancers; and preservatives. The coacervate composition isprovided in a suitable vessel for storage, distribution and/or use ofthe composition. In one aspect, the coacervate composition is providedin a tube, a medical syringe, an IV bag. In another aspect, thecoacervate composition is delivered to a patient in an amount effectiveto treat a myocardial infarction, for example by direct injection of thecoacervate composition comprising IL-10 and FGF-2 into the heart, e.g.,the myocardium at or adjacent to an infarct.

An “amount effective” for treatment of a condition is an amount of anactive agent or dosage form, such as the coacervate compositiondescribed herein, effective to achieve a determinable end-point. The“amount effective” is preferably safe—at least to the extent thebenefits of treatment outweighs the detriments and/or the detriments areacceptable to one of ordinary skill and/or to an appropriate regulatoryagency, such as the U.S. Food and Drug Administration. In the context ofcancers, the end point may be increased survival, reduction in tumormass, or any other objectively-determinable indicator of improvement ina patient's condition or symptoms. Using the teachings of the presentdisclosure, a person of ordinary skill in the arts can prepare thecoacervate composition described herein, and titrate the effect on anyobjectively-determinable end-point, such as tumor mass or survival, forinstance first in an animal model and later in humans. As shown in theExamples below, an example of an “amount effective” is indicated.

The coacervate composition may be administered continually for a periodof time, or at intervals, ranging from hourly, weekly, monthly, oryearly, including increments therebetween, such as from one to six timesper day, daily, every other day, weekly, bi-weekly, monthly, bi-monthly,quarterly, etc. An appropriate dosing schedule can be determined by aperson of ordinary skill, such as a physician, and can also be tailoredto disease progression and severity in a patient (e.g., staging and/orgrading) and/or the type of cancer, or improvement in cardiac output orrepair.

In use, according to one aspect, the coacervate composition is deliveredto a patient in an amount effective to treat a cancer or hyperplasia ina patient. Cancers or hyperplasia particularly suited for treatment inthis manner include solid tumors, that is, a mass or masses of cancerouscells, such as melanoma, or other hyperplasia. The composition isdelivered, for example by injection, at or adjacent to a mass. In oneaspect, the composition is delivered to a patient at or adjacent to amass, such as a tumor, the composition comprising IL-12. The compositionis administered in an amount effective to treat the cancer, that is toimprove one or more clinically-relevant markers, such as to reduce masssize, to destroy the mass, to reduce the cancer grade, and/or to improvepatient survival.

In another aspect, the coacervate composition is delivered to a patientin an amount effective to treat a cardiovascular disease, such ascoronary heart disease, including treatment of ischemic conditions, suchas myocardial infarction. In one aspect, the composition is delivered toa patient's myocardium at or adjacent to an infarct, the compositioncomprising an antiinflammatory immunomodulatory cytokine, such as IL-10,and an amount of an angiogenic growth factor, such as FGF-2. Thecytokine and angiogenic growth factor are administered in an amounteffective to treat the infarct, that is to improve one or moreclinically-relevant markers, such as to improve cardiac functionparameters such as myocardial elasticity, to reduce infarct size, toincrease revascularization of the infarct, to reduce scarring of themyocardium, and/or stimulate repair of the myocardium. Other conditions,such as myocardial reperfusion injury and peripheral artery disease maybe treated in the same manner.

Example 1—Synthesis and Testing of PEAD

Synthesis and testing of PEAD, PEAD-heparin, and PEAD FGF2 are describedin U.S. Pat. No. 9,023,972, which is incorporated by reference in itsentirety. Briefly, for synthesis of PEAD—t-BOC protected aspartic acid(t-BOC Asp), t-BOC protected arginine (t-BOC-Arg) (EMD Chemicals, NJ),ethylene glycol diglycidyl ether (EGDE), trifluoroacetic acid (TFA) (TCIAmerica, OR), anhydrous 1,4-dioxane and tetra-n-butylammonium bromide(TBAB) (Acros organics, Geel, Belgium), dicyclohexylcarbodiimide (DCC),N-hydroxysuccinimide (NHS) (Alfa Aesar, MA) and 4-dimethylaminopyridine(DMAP) (Avocado Research Chemicals Ltd, Lancaster, UK) were used forPEAD synthesis without purification. The synthesis of PEAD is performedas follows. EGDE and t-BOC Asp were polymerized in 1,4-dioxane under thecatalysis of TBAB. t-BOC protection was later removed by TFA to generateprimary amine. t-BOC-Arg was conjugated by DCC/NHS/DMAP couplingfollowed by the second de-protection to yield PEAD. The chemicalstructure was confirmed using NMR and FT-IR. The molecular weight ofPEAD was measured by PL-GPC 50 Plus-RI equipped with a PD 2020 lightscattering detector (Varian, MA). Two MesoPore 300×7.5 mm columns and0.1% of LiBr in DMF were used as solid phase and mobile phase,respectively. In one example, the weight-average molecular weight (Mw)is 30,337 Da with polydispersity index (PDI) 2.28.

Since PEAD is a positively-charged molecule, addition of PEAD intoheparin solution should neutralize the negative charge of heparin andforms PEAD/heparin complex. To test the binding ability of PEAD toheparin, zeta potential measurement was performed and the zeta potentialof the complex shifted from negatively-charged (−45 mV) at ratio 1 topositively-charged (+23.2 mV) at ratio 10. Continuing adding more PEADdid not change the zeta potential and +23.2 mV is close to the zetapotential of PEAD itself. Data suggested that for the described PEADpreparation after ratio 10 the complex was all covered by PEAD. Besidesit also shows at ratio 5 PEAD almost neutralized all negative charges ofheparin. From the macroscopic observation, below ratio 5 the addition ofPEAD let the heparin solution became more turbid and precipitate wasseen after a few minutes. Whereas the ratio was over 5, the addition ofPEAD would let the solution become clear again.

Further confirming the binding ability, different amounts of PEAD toheparin solutions were mixed and then precipitated by centrifugation.Because the neutralization of the negative-charged heparin favors theformation of precipitate, we measured the amount of heparin left in thesupernatant was measured to determine the binding affinity between PEADand heparin. For this assay, a heparin binding dye, dimethylmethyleneblue (DMB) was applied to detect free heparin by measuring theabsorption of DMB at 520 nm. The result shows the amount of heparin inthe supernatant was gradually lowered with the addition of PEAD. Whenthe ratio of PEAD to heparin is over 3, >90% of heparin was precipitatedthrough centrifugation. At the ratio 5, that would be >99% of heparin.This result has a good correlation with that of zeta potentialmeasurement because both experiments suggest at ratio 5 PEAD and heparinhas the maximum interaction.

It is understood that a variety of growth factors can bind to heparinwith the dissociation constant (Kd) from μM to nM. The loadingefficiency of growth factors to PEAD/heparin complex was studied. 100 ngor 500 ng of fibroblast growth factor-2 (FGF-2) plus ¹²⁵I-labeled FGF-2used as a tracer were mixed with heparin then added into PEAD solution.After staying at room temperature for 2 hr, centrifugation was used toprecipitate PEAD/heparin/FGF-2. The amount of unloaded FGF-2 remainingin the supernatant can be determined by a gamma counter. The resultshowed PEAD/heparin loaded˜68% of FGF-2 for both high and low amounts ofFGF-2. The other growth factor, NGF, the release is faster. The initialburst reached almost 20%. The release sustained till day 20 and reacheda plateau corresponding to ˜30% of the loaded NGF.

Example 2—Cytokine Delivery and Treatment of Cancer

The following represents use of the compositions described herein in thecancer setting using coacervates integrating the immunostimulatorycytokines IL-2, IL-12, and IFN-γ as locoregional therapies againstmurine melanomas.

Methods:

Generation of QC of Coacervates: Coacervates containing cytokines willbe prepared according to the following. Briefly, poly(ethyleneargininylaspartate diglyceride) (PEAD) and clinical-grade heparin(Scientific Protein Labs, Waunakee, Wis., USA) will be separatelydissolved in 0.9% normal saline (Baxter Healthcare, Deerfield, Ill.,USA) at 10 mg/ml and sterilized by passing through a 0.22 μm syringefilter. A 5:1 ratio of PEAD and heparin by weight will be used tomaintain electric neutrality (that is, the coacervate has a neutralcharge where the ratio of the polycationic polymer to the polyanionicpolymer is such that the overall positive charge of the polycationicpolymer equals or approximates the overall negative charge of thepolyanionic polymer in the coacervate). Heparin will be first complexedwith a pre-determined, equal amount of recombinant murine IL-2,IL-12p70, or IFN-γ (all from PeproTech, Rocky Hill, N.J., USA) and mixedwell. PEAD will subsequently be added into the solution containing[heparin:cytokine] complexes. Self-assembly of PEAD and[heparin:cytokine] will immediately precipitated the ternary complex outof solution to form the cytokine coacervates. Precipitation ofcoacervate complexes will immediately increase turbidity in solution.Coacervates will be freshly-prepared immediately before all in vitro andin vivo experiments to avoid aggregation. Coacervate droplet sizes willbe measured using a Zetasizer Nano ZS90 (Malvern, Worcestershire, UK)and reported as the mean with polydispersity index (PDI) from 25measurements. Results will then be averaged from measurements of threeindependent coacervate samples for each cytokine cohort. PDI in the areaof light scattering will depict the droplet size distribution.

The cytokine release profile of the prepared coacervates will bedetermined in vitro as previously described (Chen W C, et al.,Controlled dual delivery of fibroblast growth factor-2 andInterleukin-10 by heparin-based coacervate synergistically enhancesischemic heart repair. Biomaterials. 2015; 72:138-51. PMID: 26370927;PMCID: PMC4617784). To simulate release of cargo molecules,phosphate-buffered saline (PBS) supplemented with 0.5 U/mL heparinase IIwill be added to each sample to bring up the final volume to 200 μL.Four independent samples were then placed statically in a humidifiedcell culture incubator at 37° C. At Day 0, 0.5, 1, 4, 7, 10, 14, and 21,samples will be pelleted by centrifugation (12,100 g for 10 min),followed by the collection of supernatants. Samples will then bereplenished with fresh solution and well mixed before being returned tothe incubator. Solutions will be stored at −80° C. prior to analysisusing cytokine-specific ELISA (BD Biosciences).

Tumor Therapy Experiments: C57BL/6 mice will be injected s.c. in theirright flank with 1-2×10⁵ syngeneic melanoma cells (BRAF^(WT) B16 orBRAF^(V600)E BP) and tumors allowed to establish for 7-10 days.Tumor-bearing mice will then be randomized into cohorts of 5 mice/group,with each group exhibiting similar mean tumor sizes (based on theproduct of orthogonal measurements in mm²). Cohorts of mice will then beinjected intra-tumorally (i.t.) with 50 microliters of i.) PBS(control), ii.) a cytokine-free coacervate (control), iii.) coacervatescontaining rmIL-2, iv.) coacervates containing rmIL-12, v.) coacervatescontaining rmIFN-γ, vi.) rmIFN-γ (control), vii.) rmIL-2 (control),viii.) rmIL-12 (control), and/or ix.) rmIFN-γ (control). Combinations ofcytokines also are tested essentially as indicated above. If in vitro QCanalyses suggest abbreviated release of a given incorporated cytokine,individual cohorts of mice may be retreated with an identical i.t.injection (PBS, control coacervate or cytokine-containing coacervate)based on the kinetic profile of cytokine release. Mice will be monitoredfor tumor size over time, as well as, time-to-euthanasia as a measure ofsurvival. Animals will be euthanized if melanomas exceed a size of 400mm² or if they become openly ulcerated. It is to be expected thatanimals undergoing a protective immune response will exhibitinflammation at tumor sites, hence the reddening of lesions may bereflective of an ongoing local immune response and will not be groundsfor euthanasia. Mice will also be euthanized if they exhibit signs ofdiscomfort or behavioral abnormalities (i.e. hunching, laboredbreathing, fur ruffling), or if they exhibit a >20% weight-loss onprotocol. Experiments will be performed at least twice for both the B16and BP melanoma models.

Second-level analyses of coacervates that mediatestatistically-significant therapeutic benefits to melanoma-bearing micewill be evaluated in 2-site (s.c. right flank+s.c. left flank) melanomamodels, where only tumor on the right flank will be treated by i.t.delivered coacervates (vs. PBS). This will allow us to discern systemicimmune benefits resulting from treatment on both directly-treated tumors(right flank) vs. untreated lesions (left flank), allowing us tointerpret therapy efficacy against disseminated disease. At time ofeuthanasia, we will harvest the lungs of tumor-bearing mice to enumeratepulmonary metastases (i.e. both B16 and BP melanomas spontaneouslymetastasize to the lungs) as we have previously described³⁶. If >1cytokine-containing coacervate mediates anti-melanoma efficacy in vivo,we plan to determine whether combination of such species is capable ofproviding improved treatment outcome. Such studies would involve cohorts(n=5 each) of melanoma-bearing animals treated with i.) PBS, ii.)cytokine-free coacervate, iii.) cytokine 1 coacervate, iv.) cytokine 2coacervate, v.) cytokine 1 coacervate+cytokine 2 coacervate.Combinations of cytokines also are tested essentially as indicatedabove.

In both single and 2-site melanoma models we may include additionalanimals per cohort to allow for immune monitoring. In particular, 1-2additional mice/treatment cohort would allow for us to harvest spleens,tumor-draining lymph nodes (TDLNs) and tumors for analysis ofanti-melanoma CD8⁺ T cell frequencies (after stimulation with melanomaantigen [MART1, gp100, TRP2]-derived peptides as monitored in IFN-γELISPOT assays) and total lymphocyte subset counts (i.e. CD4⁺ T cells,CD8⁺ T cells, NKp46⁺ NK cells, CD4⁺Foxp3⁺ Treg, and CD11b⁺Gr1⁺ MDSC asdetermined by flow cytometry). It would be anticipated thatcytokine-containing coacervates mediating treatment benefits wouldpromote increased anti-specific CD8⁺ T cell frequencies and reducedpresence of Treg/MDSC based on our past experience with effectiveimmunotherapies in murine melanoma models.

Statistics: Non-parametric tests will be used for the comparison ofdifferent groups of in vitro experiments. Mixed effect models will befit to the log scale tumor volume to compare the growth curve ofdifferent treatment groups to controls. Time-to-euthanasia will besummarized by the Kaplan-Meier method, and log-rank tests will be usedto compare the survival curves between different treatment groups.

Example 3—Cytokine Delivery and Treatment of Myocardial Infarction (MI)

Myocardial infarction (MI) causes myocardial necrosis, triggers chronicinflammatory responses, and leads to pathological remodeling. Controlleddelivery of a combination of angiogenic and immunoregulatory proteinsmay be a promising therapeutic approach for MI. We investigated thebioactivity and therapeutic potential of an injectable, heparin-basedcoacervate co-delivering an angiogenic factor, fibroblast growthfactor-2 (FGF2), and an anti-inflammatory cytokine, Interleukin-10(IL-10) in a spatially and temporally controlled manner. Coacervatedelivery of FGF2 and IL-10 preserved their bioactivities on cardiacstromal cell proliferation in vitro. Upon intramyocardial injection intoa mouse MI model, echocardiography revealed that FGF2/IL-10 coacervatetreated groups showed significantly improved long-term LV contractilefunction and ameliorated LV dilatation. FGF2/IL-10 coacervatesubstantially augmented LV myocardial elasticity. Additionally,FGF2/IL-10 coacervate notably enhanced long-term revascularization,especially at the infarct area. In addition, coacervate loaded with 500ng FGF2 and 500 ng IL-10 significantly reduced LV fibrosis, considerablypreserved infarct wall thickness, and markedly inhibited chronicinflammation at the infarct area. These results indicate that FGF2/IL-10coacervate has notably greater therapeutic potential than coacervatecontaining only FGF2. Overall, our data suggest therapeuticallysynergistic effects of FGF-2/IL-10 coacervate, particularly coacervatewith FGF2 and 500 ng IL-10, for the treatment of ischemic heart disease.

Example 4—Controlled Dual Delivery of Fibroblast Growth Factor-2 andInterleukin-10 by Heparin-Based Coacervate Synergistically EnhancesIschemic Heart Repair

We recently developed a controlled delivery system that utilizes thecharge interaction between a biodegradable polycation, poly(ethyleneargininylaspartate diglyceride) (PEAD), and a natural polyanion,heparin, to form coacervate. This heparin-based coacervate deliveryplatform protects and steadily releases heparin-binding growth factors,including fibroblast growth factor-2 (FGF2) (See, e.g., U.S. Pat. No.9,023,972, which is incorporated herein by reference in its entirety),nerve growth factor (NGF), heparin-binding epidermal growth factor-likegrowth factor (HB-EGF), stromal cell-derived factor (SDF)-1α, and bonemorphogenetic protein-2 (Chu, H., et al., A [polycation:heparin] complexreleases growth factors with enhanced bioactivity. Journal of ControlledRelease, 2011. 150(2): p. 157-163; Johnson, N. R. and Wang, Y.,Controlled delivery of heparin-binding EGF-like growth factor yieldsfast and comprehensive wound healing. Journal of Controlled Release,2013. 166(2): p. 124-129; Li, H., et al., Sustained Release of BoneMorphogenetic Protein 2 via Coacervate Improves the Osteogenic Potentialof Muscle-Derived Stem Cells. Stem Cells Translational Medicine, 2013.2(9): p. 667-77; and Li, H., et al., Sustained Release of BoneMorphogenetic Protein 2 via Coacervate Improves the Osteogenic Potentialof Muscle-Derived Stem Cells. Stem Cells Translational Medicine, 2013.2(9): p. 667-77). In addition, heparin-based coacervate has been shownto efficiently deliver HB-EGF in a mouse model of skin wound healing,accelerating keratinocyte migration and wound closure, and FGF2 in amouse model of subcutaneous injection, promoting local neoangiogenesisand blood vessel maturation (Johnson, N. R. and Wang, Y., Journal ofControlled Release, 2013. 166(2): p. 124-129 and Chu, H., et al.,Injectable fibroblast growth factor-2 coacervate for persistentangiogenesis. Proceedings of the National Academy of Sciences, 2011.108(33): p. 13444-13449. Furthermore, in a murine MI model, coacervatecontaining 500 ng FGF2 has been proven effective in augmentingfunctional angiogenesis and blood vessel stabilization, reducingcardiomyocyte death and peri-infarct fibrosis, and improving cardiacfunction (Chu, H., et al., The effect of a heparin-based coacervate offibroblast growth factor-2 on scarring in the infarcted myocardium.Biomaterials, 2013. 34(6): p. 1747-1756). Utilizing the versatileprotein-binding capacity of heparin, we theorized that dual delivery ofFGF2 and an anti-inflammatory agent by coacervate can be therapeuticallymore effective than the delivery of FGF2 alone.

Interleukin-10 (IL-10) is a pleiotropic cytokine that exhibits broadimmunoregulatory and anti-inflammatory activities. Human IL-10 binds toheparin with high affinity at pH 7.4 (Kd=54±7 nM). The role of IL-10 inthe cardiac milieu has been investigated in recent years. In congestiveHF patients, higher plasma levels of anti-inflammatory mediators such asIL-10 notably correlates with augmented contractile function of the leftventricle (LV). Daily subcutaneous injections of recombinant human IL-10(rhIL-10, 75 μg/kg-day) for 4 weeks in a rat model of acute MI (AMI)resulted in significantly reduced productions of proinflammatorycytokines, diminished myocardial macrophage infiltration, and augmentedLV function (Stumpf, C., et al., Interleukin-10 improves leftventricular function in rats with heart failure subsequent to myocardialinfarction. European Journal of Heart Failure, 2008. 10(8): p. 733-739).Nonetheless, due to its short half-life (2.7 to 4.5 hours) aftersubcutaneous injection, it typically requires repeated administrationsof high-dose IL-10 to achieve therapeutic potency, leading to increasingrisks of side-effects and high treatment cost. Given its highheparin-binding affinity, coacervate may serve as an ideal vehicle forsustained, localized delivery of IL-10 and further reduce the requiredtherapeutic dosage.

Controlled co-delivery of two trophic factors to promote tissue repairhas lately been explored. In particular, sustained delivery of FGF2 andhepatocyte growth factor (HGF) via cross-linked albumin-alginatemicrocapsules augmented angiogenic and arteriogenic responses, improvedcardiac perfusion and function, and attenuated cardiac hypertrophy andfibrosis (Banquet, S., et al., Arteriogenic Therapy by IntramyocardialSustained Delivery of a Novel Growth Factor Combination Prevents ChronicHeart Failure. Circulation, 2011. 124(9): p. 1059-1069). Co-delivery ofangiogenic FGF-2 and arteriogenic platelet-derived growth factor(PDGF)-BB with self-assembling peptides resulted in reduced infarctsize, stable vessel formation, and improvement of cardiac function (Kim,J. H., et al., The enhancement of mature vessel formation and cardiacfunction in infarcted hearts using dual growth factor delivery withself-assembling peptides. Biomaterials, 2011. 32(26): p. 6080-6088).Using a poly(D,L-lactic-co-glycolic acid) microsphere/alginate hydrogelhybrid system, combined delivery of vascular endothelial growth factor(VEGF) and angiopoietin-1 synergistically enhanced vascular maturationand attenuated muscle degeneration at the ischemic site in an murinemodel hind-limb ischemia, more effective than single factor delivery(Shin, S.-H., et al., Co-delivery of Vascular Endothelial Growth Factorand Angiopoietin-1 Using Injectable Microsphere/Hydrogel Hybrid Systemsfor Therapeutic Angiogenesis. Pharmaceutical Research, 2013. 30(8): p.2157-2165). On the other hand, our group recently demonstrated thatheparin-based coacervate is capable of incorporating and sustaining therelease of VEGF and HGF for at least three weeks (Awada, H. K., Johnson,N. R., and Wang, Y., Dual Delivery of Vascular Endothelial Growth Factorand Hepatocyte Growth Factor Coacervate Displays Strong AngiogenicEffects. Macromolecular Bioscience, 2014. 14(5): p. 679-686). Dualdelivery of VEGF and HGF by coacervate showed stronger angiogeniceffects on endothelial cell proliferation and tube formation in vitrothan free or coacervate delivery of individual factor (Awada, H. K.,Johnson, N. R., and Wang, Y., Macromolecular Bioscience, 2014. 14(5): p.679-686).

It was hypothesize that dual delivery of FGF2 and IL-10 synergisticallyenhances their angiogenic and/or cardioprotective potency in theischemic heart. Here, the characterized FGF2/IL-10 coacervate ischaracterized and its bioactivity on cardiac stromal cells in vitro isinvestigated. The therapeutic efficacy of FGF2/IL-10 coacervate wasevaluated in a mouse AMI model. The data suggest that controlled releaseof FGF2 and IL-10 by heparin-based coacervate exerts synergistic effectsin improving long-term cardiac function, augmenting myocardialelasticity, promoting revascularization, ameliorating myocardialfibrosis, and inhibiting chronic inflammation.

Material and Methods

Preparation of FGF-2/IL-10 Coacervate: Poly(ethylene argininylaspartatediglyceride) (PEAD) was synthesized as previously described. PEAD andclinical-grade heparin (Scientific Protein Labs, Waunakee, Wis., USA)were separately dissolved in 0.9% normal saline (Baxter Healthcare,Deerfield, Ill., USA) at 10 mg ml⁻¹ and sterilized by passing through0.22 μm syringe filter. A 5:1 ratio of PEAD and heparin by weight wasused to maintain electric neutrality as previously described [7].Heparin was first complexed with a pre-determined, equal amount ofrecombinant human FGF-2 (rhFGF-2; 17.2 kDa protein consisting of 154amino acid residues) and IL-10 (rhIL-10; 18.6 kDa protein of consistingof 161 amino acid residues) (both from PeproTech, Rocky Hill, N.J., USA)and mixed well. PEAD was subsequently added into the solution containing[heparin:FGF-2/IL-10] complexes. Self-assembly of PEAD and[heparin:FGF-2/IL-10] immediately precipitated the ternary complex outof solution to form the FGF-2/IL-10 coacervate. Precipitation ofcoacervate complexes immediately increased opaque turbidity in solution.Coacervate was freshly prepared immediately before all in vitro and invivo experiments to avoid aggregation. Coacervate droplet size wasmeasured by Zetasizer Nano ZS90 (Malvern, Worcestershire, UK) andreported as the mean with polydispersity index (PDI) from 25measurements. Results were then averaged from measurements of threeindependent coacervate samples. PDI in the area of light scatteringdepicts the droplet size distribution.

Fluorescent labeling of FGF-2/IL-10 Coacervate: To fluorescentlyvisualize the incorporation of FGF2 and IL-10 in coacervate complexes,amine-reactive dyes (Thermo Scientific, Waltham, Mass., USA) wereutilized to label FGF2 and IL-10 molecules, following the manufacturer'sinstructions. Briefly, FGF2 and IL-10 solutions were added into vialscontaining concentrated NHS ester-activated derivatives of DyLight 488and DyLight 594 respectively and reacted at room temperature for 1 hour.A spin desalting column was used to remove unreacted dyes. To triplylabel biological components in FGF-2/IL-10 coacervate, FGF2 and IL10were first labelled with NHS-DyLight 488 and NHS-DyLight 405individually. FGF2-DL488 and IL-10-DL405 were then mixed well withheparin before rhodamine conjugated ulex europaeus agglutinin I (UEA-1)was applied to label heparin. PEAD was then added into the solutioncontaining [heparin-rhodamine: FGF2-DL488/IL-10-DL405] complexes to formcoacervate.

In vitro Release Profile of FGF-2/IL-10 Coacervate: The release profileof FGF2/IL-10 coacervate was determined in vitro as previouslydescribed. Briefly, FGF2/IL-10 coacervate was freshly prepared with amass ratio of PEAD:heparin:FGF2:IL-10=500:100:1:1, using 100 ng each ofFGF2 and IL-10. To simulate release of cargo molecules in vivo,additional phosphate-buffered saline (PBS) supplemented with 0.5 U/mLheparinase II was added to each sample to bring up the final volume to200 μL. Four independent samples were then placed statically in ahumidified cell culture incubator at 37° C. At Day 0, 0.5, 1, 4, 7, 10,14, and 21, samples were pelleted by centrifugation (12,100 g for 10min), followed by the collection of supernatants. Samples were thenreplenished with fresh solution and well mixed before being returned tothe incubator. Solutions were stored at −80° C. for future analysis.After the final collection on Day 21, samples were replenished with PBSsupplemented with 2 U/mL heparinase II and incubated at 37° C. overnightin order to dissociate the remaining coacervate. The amount of FGF2 andIL-10 released into the supernatant was quantified by enzyme-linkedimmunosorbent assay (ELISA) for FGF2 or IL-10 respectively (both fromAbcam, Cambridge, Mass., USA), following the manufacturer'sinstructions. Supernatants collected from four samples at all-timepoints were analyzed simultaneously. The absorbance was recorded bySynergyMX (Biotek, Winooski, Vt., USA) or Infinite 200 PRO plate reader(Tecan, Mannedorf, Switzerland). Results were averaged. The loadingefficiency was determined from the first collection immediately afterthe initial resuspension (Day 0).

Primary Cell Isolation and Culture: Single donor-derived human umbilicalvein endothelial cells (HUVECs) and human cardiac fibroblasts (hCFs)were purchased from Lonza (Allendale, N.J., USA) and respectivelyexpanded in complete endothelial cell growth medium 2 (EGM-2, Lonza) andDMEM high glucose supplemented with 10% fetal bovine serum (FBS) and 1%penicillin-streptomycin (P/S) (all from Life Technologies, Grand Island,N.Y., USA). Mouse cardiac fibroblasts (mCFs) were isolated as previouslydescribed (Balasubramanian, S., et al., 03 Integrin in CardiacFibroblast Is Critical for Extracellular Matrix Accumulation duringPressure Overload Hypertrophy in Mouse. PLoS ONE, 2012. 7(9): p. e45076)and expanded in DMEM high glucose with 10% FBS and 1% P/S. Human heartpericytes (hHPs) were isolated and purified by flow cytometry as wepreviously reported (Chen, W. C. W., et al., Human Myocardial Pericytes:Multipotent Mesodermal Precursors Exhibiting Cardiac Specificity. STEMCELLS, 2015. 33(2): p. 557-573). hHPs were expanded in DMEM high glucosewith 20% FBS and 1% P/S. Primary cells at passage 5-7 were used insubsequent experiments.

Measurement of Cell Proliferation in vitro: HUVECs, hCFs, hHPs, and mCFswere trypsinized and plated in triplicate (1.5×10³ cells/well) overnightwith 100 μl complete culture media in bottom wells of a HTS transwell-96well permeable support system (Corning, Tewksbury, MA, USA). Immediatelybefore the transwell support was assembled, all bottom wells were firstfilled up with 135 μl fresh serum-free basal media (EBM-2 for HUVECs andDMEM for hCFs, hHPs, and mCFs; both supplemented with 1% P/S) with orwithout 10 ng/ml (for HUVECs) or 100 ng/ml (for all other cell types) oftumor necrosis factor alpha (TNF-α). Free 500 ng FGF2 combined witheither 100 ng or 500 ng IL-10 and coacervate containing a fixed load of500 ng FGF2 alone or combined with either 100 ng or 500 ng IL-10 wereresuspended in 75 μl serum-free basal media and then added intotranswells. Control transwells were added with plain basal media with orwithout empty coacervate vehicle. The final concentration of serum wasapproximately 33% of that in complete culture media in each well. Plateswere assembled and subsequently incubated for 72 hours under ambientconditions. After washing all wells, CellTiter 96© AQueous One SolutionCell Proliferation Assay (MTS) reagent (Promega, Madison, Wis., USA) inDMEM was added. The plate was incubated in 5% CO₂ at 37° C. for 3 hrs,at which point the absorbance at 490 nm (with reference at 650 nm) wasread with Infinite 200 PRO plate reader (Tecan, Mannedorf, Switzerland).All experiments were independently repeated 3 times. Results wereindividually normalized to each experimental control and then averaged.

Experimental Animals: A Total of 77 male BALB/cJ mice at 9-12 weeks old(Jackson Laboratory, Bar harbor, ME, USA) were used for this study.

Intramyocardial Administration of FGF-2/IL-10 Coacervate in a MouseModel of Acute Myocardial Infarction (AMI): After the induction ofanesthesia with 4% isoflurane gas, mice were intubated andinhalationally anesthetized with 2% isoflurane gas throughout thesurgery. The induction of myocardial infarction (MI) and intramyocardialinjection have been performed as previously reported (Chu, H., et al.,Biomaterials, 2013. 34(6): p. 1747-1756 and Chen, C.-W., et al., HumanPericytes for Ischemic Heart Repair. STEM CELLS, 2013. 31(2): p.305-316). In brief, MI was microscopically induced by permanent ligationof the left anterior descending coronary artery (LAD). Mice were thenrandomly assigned to one of the five groups: saline control, FGF2 500 ngcoacervate (Coa-F-500), Free FGF2/IL-10 500/500 ng (Free-F/I-500/500),FGF2/IL-10 500/100 ng coacervate (Coa-F/I-500/100), or FGF2/IL-10500/500 ng coacervate (Coa-F/I-500/500). Five minutes after theinduction of MI, free or coacervate FGF2/IL-10 diluted in 30 μl ofsterile 0.9% normal saline were injected at three sites of the ischemicmyocardium (center and two borders of the infarct). Control micereceived injections of 30 μl saline.

Echocardiography: Echocardiographic studies were performed repeatedlybefore surgery and at 5 days, 2 and 6 weeks post-surgery to assess thecardiac function as we previously described (Chu, H., et al.,Biomaterials, 2013. 34(6): p. 1747-1756 and Chen, C.-W., et al., STEMCELLS, 2013. 31(2): p. 305-316). Briefly, mice were anesthetized with 2%isoflurane gas and immobilized on a heated stage equipped withelectrocardiography. Heart and respiratory rates were continuouslymonitored while the body temperature was maintained at 37° C.Echocardiographic parameters were measured using a high-resolutionechocardiography system (Vevo 2100) equipped with a high-frequencylinear probe (MS400, 30 MHz) (FUJIFILM VisualSonics, Toronto, Ontario,Canada). Three hundred B-mode frames were acquired at a frame rate of 40Hz during each scan. End-systolic dimension (ESD) and end-diastolicdimensions (EDD) were determined from the short axis images of the LVand measured from 10 consecutive beats using the M-mode tracing.End-systolic area (ESA) and end-diastolic area (EDA) were measured fromshort-axis images of the LV. All echocardiographic measurements weretaken at the mid-infarct level in LV. Functional parameters, includingLV fractional shortening (LVFS), LV fractional area change (LVFAC), andLV ejection fraction (LVEF), were determined as previously described(Manning, W. J., et al., In vivo assessment of LV mass in mice usinghigh-frequency cardiac ultrasound: necropsy validation. American Journalof Physiology—Heart and Circulatory Physiology, 1994. 266(4): p.H1672-H1675; Pollick, C., Hale, S. L., and Kloner, R. A.,Echocardiographic and cardiac doppler assessment of mice. Journal of theAmerican Society of Echocardiography, 1995. 8(5, Part 1): p. 602-610;and Wandt, B., et al., Echocardiographic assessment of ejection fractionin left ventricular hypertrophy. Heart, 1999. 82(2): p. 192-198). Micedied or sacrificed for histological analysis prior to 6 weekspost-injection were not included in the echocardiographic study.

Ultrasonic Analysis of Myocardial Elasticity: The ultrasound in-phaseand quadrature (IQ) data were separately acquired at 6 weeks post-MIduring the echocardiographic scanning described in the above section(N=3 per group). The IQ data were then converted to the radio frequency(RF) data using standard quadrature sampling algorithms and subsequentlyanalyzed by a blinded investigator. Briefly, pixels were selected in thelateral (infarcted region) and anterior medial (non-infarct region)walls of LV in the first B-mode frame. The 2D phase-sensitive speckletracking was then applied to the RF data to obtain frame-to-frame axialdisplacements (direction along the ultrasound beam) of the selectedpixels (O'Donnell, M., et al., Internal displacement and strain imagingusing ultrasonic speckle tracking. Ultrasonics, Ferroelectrics, andFrequency Control, IEEE Transactions on, 1994. 41(3): p. 314-325 andLubinski, M. A., et al., Speckle tracking methods for ultrasonicelasticity imaging using short-time correlation. Ultrasonics,Ferroelectrics, and Frequency Control, IEEE Transactions on, 1999.46(1): p. 82-96). Axial displacements were accumulated during eachcardiac cycle (from diastole to systole). Axial strains in LV wall wereobtained by derivative of the accumulated axial displacements. Tounbiasedly estimate myocardial elasticity, two regions of interest (ROI)in the axial strain map were respectively selected in the infarcted andnon-infarct LV walls. Axial strains in these ROIs were spatiallyaveraged and then normalized by dividing the averaged strain of theinfarcted ROI by that of the non-infarct ROI.

Histology and Immunohistochemistry: Mice were sacrificed at 6 weekspost-surgery. Intraventricular injection of 1M potassium chloride (KCl)was performed to arrest hearts in diastole. For histology andimmunohistochemistry, harvested hearts were flash frozen in2-methylbutane (Sigma-Aldrich, St. Louis, Mo., USA) pre-cooled in liquidnitrogen, preserved at −80° C., and then processed as formerly described(Chu, H., et al., Biomaterials, 2013. 34(6): p. 1747-1756 and Chen,C.-W., et al., STEM CELLS, 2013. 31(2): p. 305-316). Briefly, frozenhearts were serially cryosectioned at 6-8 μm thickness from apex to theligation level (approximately 0.5 mm in length). Each series contained18-21 heart sections and was collected on one glass slide. Hematoxylinand eosin (H&E) staining was performed following the standard protocol.For immunohistochemistry, sections were fixed in a pre-cooled (−20° C.)mixture of methanol and acetone (1:1) for 5 min or in 4%paraformaldehyde for 8 min prior to staining. Non-specific antibodybinding was blocked with 10% donkey or goat serum for 1-2 hours at roomtemperature (RT), and, if necessary, with the Mouse-on-Mouse antibodystaining kit (Vector Laboratories, Burlingame, Calif., USA). Sectionswere incubated overnight at 4° C. with the following primary antibodies(all diluted with 5% donkey or goat serum in PBS): rat anti-mouse CD31antibody (diluted at 1:100; Becton-Dickinson Biosciences, FranklinLakes, N.J., USA), mouse anti-mammalian alpha-smooth muscle actin(αSMA)-FITC (diluted at 1:100; Sigma-Aldrich, St. Louis, Mo., USA),and/or rat anti-mouse CD68 antibody (diluted at 1:200; Abcam, Cambridge,Mass., USA). Sections were then incubated at RT for 1 hour with thefollowing fluorochrome-conjugated antibodies: donkey anti-rat-Alexa594IgG or goat anti-rat-Alexa488 IgG (both diluted at 1:250; JacksonLaboratory, Bar Harbor, Me., USA). Nuclei were stained with4′,6-diamidino-2-phenylindole (DAPI) (1:1000, Life Technologies, GrandIsland, N.Y., USA) at RT for 5 min. Immunofluorescent images were takenby Nikon Eclipse Ti fluorescence microscope equipped with NIS-ElementsAR imaging software (both from Nikon, Tokyo, Japan).

Measurement of Cardiac Fibrosis and Infarct Wall Thickness: Masson'strichrome staining kit (IMEB, San Marcos, Calif., USA) was used toreveal collagen deposition on heart serial cross-sections, following themanufacturer's instruction. The area of collagen deposition(representing fibrosis/scar) and the area of the entire left ventricularcardiac tissue (including septal area but excluding right ventricle andvoid space in the chamber cavity) were separately measured using adigital image analyzer (Image J, National Institutes of Health,Bethesda, Md., USA). Fibrotic area fraction was estimated as the ratioof left ventricular fibrotic tissue to the entire left ventriculartissue. Results were averaged from 6 randomly selected sections atcomparable infarct levels per heart. Left ventricular wall thickness atthe center of the infarct was estimated as the mean of 3 adjacentmeasurements (0.25 mm apart) and was averaged from 6 randomly selectedsections at comparable infarct levels per heart.

Quantification of Chronic Inflammation and Revascularization: Toevaluate chronic inflammation within the infarct region,immunofluorescent staining of phagocytic cell marker CD68 was performedon serial cryosections as described above. The infiltration index,represented by the number of CD68+ phagocytic cells per mm², wassubsequently computed by a blinded investigator from 6-8 randomlyselected images of the infarct region of each heart at the mid-infarctlevel, using Image J. To quantify revascularization post-MI,immunofluorescent staining of endothelial cell (EC) marker CD31 andvascular smooth muscle cell (VSMC) marker αSMA was sequentiallyperformed on serial cryosections. The capillary density, represented bythe number of CD31+ capillary ECs per mm², was subsequently computed bya blinded investigator from 6 randomly selected images of the infarct orperi-infarct area of each heart at the mid-infarct level, using Image Jas described previously (Chu, H., et al., Biomaterials, 2013. 34(6): p.1747-1756 and Chen, C.-W., et al., STEM CELLS, 2013. 31(2): p. 305-316).The VSMC density, represented by the number of perivascular (i.e.adjacent to CD31+ ECs and/or surrounding vascular structures) αSMA+cells per mm², was subsequently computed from 6 randomly selected imagesof the infarct region or peri-infarct area of each heart at themid-infarct level, using Image J.

Multi-photon Excitation Imaging: For multi-photon excitation (MPE)imaging, rhodamine tagged with UEA-1 (2 μg) was mixed well with heparinbefore PEAD was added into the solution containing [heparin:rhodamine]complexes to form coacervate. Intramyocardial injection of free orheparin-bound rhodamine-UEA-1 (2 μg) or rhodamine-UEA-1 coacervate (alldiluted in 30 μl of sterile 0.9% normal saline) was performed after theinduction of MI as described above. Hearts were harvested at 5, 14, and28 days post-injection, washed 3 times in PBS, and immediately fixed infresh 4% paraformaldehyde overnight. Hearts were then washed in PBStwice and subsequently immersed in ScaleView-A2 optical clearing agent(Olympus Scientific Solutions Americas, Waltham, Mass., USA) at 4° C.for 7-10 days. Processed hearts were block-sectioned at 1 mm thicknessto obtain cross-sections from apex to ligature immediately beforeperforming MPE imaging on an Olympus multiphoton microscope at theCenter for Biologic Imaging, University of Pittsburgh.

Statistical Analysis: All measured data are presented as mean±standarddeviation (SD). Kaplan-Meier survival curve estimation with log-ranktest was performed to compare the animal survival rate between treatmentgroups. Statistical differences between groups were analyzed by one-wayANOVA (multiple groups) or two-way repeated ANOVA (for repeatedechocardiographic measurements) with 95% confidence interval.Statistical significance was set at p≤0.05. Bonferroni multiplecomparison test was performed for ANOVA post-hoc analysis. Statisticalanalyses were performed with SigmaStat 3.5 (Systat Software) and SPSS21(IBM) statistics software.

Results

Characterization of FGF-2/IL-10 coacervate: FGF-2 and IL-10 both havehigh heparin-binding affinity (FGF-2: K_(d)≈74 nM [34]; IL-10: K_(d)≈54nM). A mixture of FGF2 and IL-10 is first complexed with heparin andsubsequently incorporated into the ternary [PEAD:heparin:FGF2/IL-10]coacervate droplets by adding PEAD. We have theorized that the fourstructural components of FGF-2/IL-10 coacervate (PEAD, heparin, FGF2,and IL-10) are evenly distributed when the coacervate forms, followingaffinity-based binding of FGF2 and IL-10 to heparin, charge interactionsbetween PEAD and heparin, and physical entrapment of FGF2 and IL-10within the complex coacervate (FIG. 1A). FGF2/IL-10 coacervate dropletshad an average size of 432.6±42.1 nm in diameter, smaller than the sizesof coacervate droplets containing only FGF2 (608.3±96.3 nm) or IL-10(502.6±101.5 nm) (FIG. 1B).

To simulate the release of cargo proteins in vivo, the amount of FGF2and IL-10 released from FGF2/IL-10 coacervate was measured by ELISAafter immersion in PBS supplemented with heparinase II (0.5 U/mL) for 0,0.5, 1, 4, 7, 10, 14, and 21 days (N=4). The loading efficiency of FGF2and IL-10 was approximately 98.0±1.6% and 97.9±0.5% respectively (FIG.1C). Cumulatively, FGF2/IL-10 coacervate released roughly 16.1±3.8% and12.5±2.4% FGF2 and IL-10 respectively during the first 12 hours andapproximately 28.7±5.0% and 14.8±2.3% respectively by 24 hours (FIG.1C). The total release of FGF2 and IL-10 from coacervate was estimatedto be 86.8±7.1% and 28.2±3.6% respectively over the 21-day duration(FIG. 1C). Final digestion with 2 U/mL heparinase II showed that atleast nearly 3% FGF2 and 15% IL-10 remained in residual coacervate.However, these data did not take into account the spontaneousdegradation of free IL-10, on average 1.59% per day, in PBS supplementedwith 0.5 U/mL heparinase II (FIG. 2).

To further demonstrate that FGF2 and IL-10 have been evenly incorporatedinto coacervate droplets, we fluorescently labeled FGF2 (DyLight 488,green in original) and IL-10 (DyLight 594, red in original). Sphericaldroplets of different sizes containing FGF-2 and IL-10 were observedfollowing coacervate formation (FIG. 1D). High-magnification confocalmicroscopy showed an even distribution of FGF-2 and IL-10 moleculeswithin a coacervate droplet (FIG. 1E). By triply labeling heparin(rhodamine, red in original FGF2 (DyLight 488, green in original) andIL-10 (DyLight 405, blue in original), the nearly homogeneous structureof FGF-2/IL-10 coacervate was further revealed (FIG. 1F).

Bioactivity of FGF-2/IL-10 coacervate in vitro: The bioactivity ofFGF2/IL-10 coacervate on cardiac stromal cell proliferation was testedin a non-contact release system to avoid direct ingestion of coacervateparticles by cells. Cells were cultured at bottom wells with treatmentsolutions in suspended transwells. Human umbilical vein endothelialcells (HUVECs), human cardiac fibroblasts (hCFs), and human heartpericytes (hHPs), and mouse cardiac fibroblasts (mCFs) were used in thisassay. Cells were seeded in complete culture media overnight and thenmaintained in diluted media throughout the experiment to simulatenutrient starvation following coronary artery blockage. Based onprevious study work, we selected a fixed load of FGF2 (500 ng) alone orcombined with a low (100 ng) or high (500 ng) load of IL-10 forcoacervate delivery (designated as Coa-F-500, Coa-F/I-500/100, andCoa-F/I-500/500 respectively). Free 500 ng FGF2 combined with either 100ng or 500 ng IL-10 served as positive controls (designated asFree-F/I-500/100 and Free-F/I-500/500 respectively). No treatment (plainor DMEM basal medium) and empty coacervate vehicle groups served asnegative controls.

After incubation for 72 hours, Free-F/I-500/100, Coa-F-500,Coa-F/I-500/100 and Coa-F/I-500/500 significantly increased HUVECproliferation when compared with the no-treatment control andFree-F/I-500/500 (FIG. 3A, all p<0.01). Coa-F/I-500/100 andCoa-F/I-500/500 showed trends of reducing hCF proliferation and notablyinhibited mCF proliferation when compared with Free-F/I-500/100 andFree-F/I-500/500 (FIG. 3A, both p<0.05). Coa-F/I-500/500 demonstratedthe most significant inhibition of mCF proliferation when compared withthe no-treatment control (p=0.006). On the other hand, Free-F/I-500/100,Free-F/I-500/500, and Coa-F/I-500/500 slightly promoted hHPproliferation (FIG. 3A, all p>0.05). No significant difference wasobserved between no-treatment control and empty coacervate vehicle groupin all four cell types (FIG. 3(A), p>0.05).

To further simulate inflammatory stress following ischemic insult, 10ng/ml of TNF-α for HUVECs and 100 ng/ml of TNF-α for all other celltypes were added into bottom wells immediately before the start of theexperiment. After incubation for 72 hours, Coa-F-500, Coa-F/I-500/100and Coa-F/I-500/500 significantly promoted HUVEC proliferation whencompared with the no-treatment control (FIG. 3(B), all p<0.05). Allthree coacervate groups exhibited reduced hCF proliferation andsignificantly inhibited mCF proliferation when compared with theno-treatment control and Free-F/I-500/100 (FIG. 3(B), all p<0.01).Similarly, Coa-F/I-500/500 showed the most striking inhibition of mCFgrowth when compared with all non-coacervate groups (all p<0.01). Alltreatment groups maintained hHP growth under inflammatory stress (allp>0.05). There was no notable difference between no-treatment and emptyvehicle groups under inflammatory stress in all tested cell populations(FIG. 3(B), p>0.05). Altogether these results suggest that FGF2/IL-10coacervate supports HUVEC growth under nutrient deprivation whileinhibiting the proliferation of CFs, especially under inflammatorystress.

Intramyocardial codelivery of FGF-2/IL-10 coacervate synergisticallyimproves cardiac function: We selected 500 ng FGF2 combined with a low(100 ng) or high (500 ng) dose of IL-10 for coacervate-based codelivery(Coa-F/I-500/100 and Coa-F/I-500/500 respectively) and examined thetherapeutic efficacy of FGF-2/IL-10 coacervate in vivo. Intramyocardialinjection of saline, coacervate containing only 500 ng FGF2 (Coa-F-500),or free FGF-2 500 ng combined with free IL-10 500 ng (Free-F/I-500/500)served as controls. The mortality rate was around 15% during andimmediately after the surgery. Among all mice which recovered from thesurgery, two died in each of the following groups: Saline,Free-F/I-500/500, and Coa-F-500, and one died in each of the followinggroups: Coa-F/I-500/100 and Coa-F/I-500/500, before the terminal timepoint. Most of these deaths occurred within the first week post-surgery.These mice were excluded from functional studies. No significantdifference in animal survival rate was noted.

Cardiac function was assessed by M- and B-mode echocardiographyperformed repeatedly at the mid-infarct level before (baseline) andafter surgery at 5 days, 2 weeks, and 6 weeks (N=8 per group exceptFree-F/I-500/500, N=7; data analyzed by two-way repeated ANOVA). Byanalyzing the treatment effect, both FGF2/IL-10 coacervate groupsexhibited substantially higher LVFS (FIG. 4A), LVFAC (FIG. 4B), and LVEF(FIG. 4C) than Saline (all p<0.001) and Free-F/I-500/500 (all p<0.05),indicating better LV contractility following controlled release of FGF2and IL-10. However, only Coa-F/I-500/500, but not Coa-F/I-500/100,showed significant improvement in all three contractile parameters whencompared with Coa-F-500 (all p<0.005). Moreover, Coa-F/I-500/500 hadsignificantly better LVFAC and LVEF than Coa-F/I-500/100 (both p<0.05),suggesting a role of IL-10 dosage in prompting a notable synergisticeffect for LV contractility.

In addition, ischemic hearts treated with either Coa-F/I-500/100 orCoa-F/I-500/500 had markedly reduced LVEDA (FIG. 4D) and LVESA (FIG. 4E)than Saline (all p<0.001) and Free-F/I-500/500 (all p<0.005), suggestingamelioration of progressive LV dilatation by FGF2/IL-10 coacervatetreatment. Similarly, only Coa-F/I-500/500, but not Coa-F/I-500/100,showed significant diminution in both dilatation parameters whencompared with Coa-F-500 (all p<0.001), suggesting a role of IL-10 dosagein ameliorating LV remodeling. Overall, our results indicate that theintramyocardial administration of FGF2/IL-10 coacervate, regardless ofthe IL-10 dose, significantly improved the LV contractile function andreduced the LV dilatation. These data further suggest the importance ofIL-10 dosage in the synergistic therapeutic effect induced by FGF2/IL-10coacervate.

FGF-2/IL-10 coacervate amends elasticity of the infarcted myocardium: Tofurther assess the effect of FGF2/IL-10 coacervate on myocardialelasticity, we performed ultrasonic strain estimation at 6 weekspost-MI. The axial strains were determined in the infarcted (area B) andnon-infarct (area A) LV walls during a cardiac cycle using the 2Dcorrelation based speckle tracking. FIG. 5A shows the axial strain mapsof normal (left panel) and untreated MI control (right panel) heartslaid over B-mode images reconstructed from IQ data respectively(negative strains in blue color and positive strains in red). Theelasticity of the infarcted myocardium was estimated from spatiallyaveraged axial strains in B and subsequently normalized by dividing theaveraged strain of B by that of A (B/A) (FIG. 5A). B-mode images ofnormal and untreated MI control hearts without strain maps and ROIs areincluded in FIG. 6 for comparison. While all groups had similar averagedaxial strains in A, Coa-F-500 (both p<0.01), Coa-F/I-500/100 (bothp<0.001), and Coa-F/I-500/500 (both p<0.001) had significantly greaternormalized strains than the saline control and Free-F/I-500/500 (FIG.4B, N=3 per group). Free-F/I-500/500 also exhibited notably highernormalized strains than the saline control (p=0.004) (FIG. 4B). Nosignificant difference was observed between the three coacervatetreatment groups (FIG. 4B, all p>0.05). These data indicate the efficacyof FGF2/IL-10 coacervate in sustaining the long-term LV myocardialelasticity.

FGF-2/IL-10 coacervate promotes long-term revascularization: The potencyof intramyocardial administration of FGF2/IL-10 coacervate on long-termrevascularization was investigated. Immunohistochemistry revealed thepresence of CD31+ endothelial cells (ECs; mostly located atmicrovasculature/capillary) (FIG. 7A) and vascular smooth muscle cells(VSMC; mostly surrounding larger blood vessels) (FIG. 7A) in the infarctand peri-infarct areas at 6 weeks post-infarction. The number of CD31+ECs was subsequently quantified in the infarct (FIG. 7B, left) andperi-infarct (FIG. 7B, right) areas (N=4 per group). Within the infarctarea, Coa-F/I-500/500 had higher CD31+EC density when compared with thesaline control (p<0.001), Free-F/I-500/500 (p=0.02), and Coa-F-500(p>0.05) (FIG. 7B, left). Coa-F/I-500/100 had higher CD31+EC densitywhen compared with the saline control (p=0.007) and Free-F/I-500/500(p>0.05) (FIG. 7B, left). Within the peri-infarct area, Coa-F/I-500/500exhibited the highest CD31+EC density when compared with the salinecontrol (p<0.001), Free-F/I-500/500 (p<0.001), Coa-F-500 (p=0.001), andCoa-F/I-500/100 (p>0.05) (FIG. 7B, right). Coa-F/I-500/100 also hadhigher CD31+EC density when compared with the saline control (p=0.008),Free-F/I-500/500 (p=0.012), and Coa-F-500 (p>0.05) (FIG. 7B, right). Thenumber of VSMCs and/or pericytes (i.e. perivascular αSMA+ cells) wasalso quantified in the infarct (FIG. 8, left) and peri-infarct (FIG. 8,right) areas (N=4 per group) at 6 weeks post-infarction. The resultsshowed that Coa-F/I-500/500 had significantly higher VSMC density whencompared with the saline control (p=0.008), Free-F/I-500/500 (p=0.038),and Coa-F-500 (p=0.031) within the infarct area (FIG. 8, left). Inaddition, hearts treated with Coa-F/I-500/100 and Coa-F/I-500/500exhibited trends of increased VSMCs within the peri-infarct area (FIG.8, right). Altogether our results suggest FGF2/IL-10 coacervatetreatment promotes long-term revascularization at 6 weekspost-infarction, especially with Coa-F/I-500/500 treatment.Additionally, these data imply that the revascularzing effect ofFGF2/IL-10 coacervate is positively correlated with the dose of IL-10.

FGF-2/IL-10 coacervate reduces myocardial fibrosis: The effect ofFGF2/IL-10 coacervate on long-term LV myocardial fibrosis was evaluatedusing Masson's trichrome histological staining (collagen depositionstained in blue/purple). At 6 weeks post-infarction, Coa-F/I-500/100 andCoa-F/I-500/500 appeared to have reduced infarct size and scar formationat the mid-infarct level when compared with the saline control andFree-F/I-500/500 (FIG. 9(A)). Quantitative analysis revealed thatCoa-F/I-500/500 exhibited notably smaller LV scar fraction than thesaline control (p=0.001) and all other treatment groups (all p>0.05)(FIG. 9(B), N=4 per group). Analysis of the LV wall thickness at theinfarct center further showed that Coa-F/I-500/500 had a significantlythicker wall than the saline control (p<0.001) and all other test groups(all p<0.05) (FIG. 9(C), N=4 per group). These data suggest the efficacyof coacervate containing higher dose of IL-10 in ameliorating theformation of myocardial fibrosis and preserving wall thicknesspost-infarction.

FGF-2/IL-10 coacervate inhibits chronic phagocytic cell infiltration: Toinvestigate the underlying mechanism for the amelioration of myocardialfibrosis, we examined the anti-inflammatory effect of FGF2/IL-10coacervate. Phagocytic cells within the infarct area were detected byanti-CD68 immunohistochemistry at 6 weeks post-infarction. All threecoacervate groups showed significantly decreased numbers of infiltratedCD68+ phagocytic cells within the infarct area when compared with thesaline control (FIG. 10, N=4 per group; Coa-F-500, p=0.037;Coa-F/I-500/100, p=0.007; Coa-F/I-500/500, p=0.002). In particular,Coa-F/I-500/100 and Coa-F/I-500/500 exhibited substantial 47.2% and59.9% reduction of CD68+ cells respectively when compared withFree-F/I-500/500 (FIG. 10, both p>0.05). Although there is nostatistical significance in the number of CD68+ cells between all threecoacervate groups, comparing with Coa-F-500, Coa-F/I-500/100 andCoa-F/I-500/500 displayed notable 23.9% and 42.3% diminution of CD68+cells respectively. Together these results suggest that coacervatedelivery of FGF2 and IL-10 increases their long-term potency forimmunoregulation, and the addition of IL-10 in FGF2 coacervate augmentsthe inhibition of chronic inflammation.

Estimation of the duration of coacervate treatment in vivo: To estimatethe duration of coacervate treatment post-MI, we employed multi-photonexcitation (MPE) imaging to detect intramyocardially injectedrhodamine-tagged coacervate (Coa-Rho). Collagen fibers were identifiedby second harmonic generation (SHG) signals. At 5 days after injection,Coa-Rho exhibited robust fluorescent signals within the infarct areawhile weak signals were detected in free (Free-Rho) or heparin-bound(Hep-Rho) rhodamine injected hearts. At 2 and 4 weeks post-MI,fluorescent signals were only detected in hearts injected with Coa-Rho,but not with Free-Rho or Hep-Rho. No signal was detected in any group at6 weeks post-MI. Quantification of the fluorescence volume found that at5 days post-injection, Coa-Rho had 28.9±11.1 and 7.1±2.7 folds highersignals than that of Free-Rho and Hep-Rho respectively (FIG. 11, N=3 pergroup, p<0.05). Moreover, when compared with the signal on Day 5,Coa-Rho had roughly 65.9% and 25.7% of residual fluorescence volume at 2and 4 weeks post-injection respectively (FIG. 11, N=3 per time point).These results suggest a temporal distribution and progressivedegradation of coacervate for at least 4 weeks in situ in infarctedhearts.

Discussion

Molecular therapy using trophic factors to promote cardiac repair andregeneration has been widely investigated. To promote revascularizationin the ischemic myocardium, angiogenic GFs such as FGF2 and VEGF havebeen successfully tested in pre-clinical models of MI. However, clinicalattempts using angiogenic GFs have demonstrated mixed results (Segers,V. M. and Lee, R., Protein Therapeutics for Cardiac Regeneration afterMyocardial Infarction. Journal of Cardiovascular Translational Research,2010. 3(5): p. 469-477). One major obstacle of molecular therapy withexogenous GFs and/or cytokines is the short in vivo half-life of mostbiological factors. In addition, the bioavailability of systemicallydelivered trophic factor(s) in the target tissue/organ variesdramatically, highly dependent on the availability of local vasculature.These shortcomings have led to common administrations of large,repetitive doses of GFs in order to achieve therapeutic efficacy, thusincreasing the risk of on-target and/or off-target side effects. Forexample, VEGF can induce nitric oxide-mediated hypotension when a doseover 50 ng/kg/min is administered by intracoronary infusions in patientswith myocardial ischemia (Henry, T. D., et al., Intracoronaryadministration of recombinant human vascular endothelial growth factorto patients with coronary artery disease. American Heart Journal, 2001.142(5): p. 872-880). To effectively augment the local bioavailabilityand potency of exogenous trophic factor(s) and minimize the requiredtherapeutic dosage in the context of ischemic insult, a suitable vehiclefor sustained, localized delivery is critically needed.

Here, nearly even incorporation and homogeneous distribution of FGF2 andIL-10 is shown within coacervate droplets. FGF2/IL-10 coacervate notonly had high loading efficiencies for FGF2 and IL-10 (approximately 98%for both) but also exhibited low initial releases of around 16.1% FGF2and 12.5% IL-10 in the presence of heparinase during the first 12 hoursand relatively linear releases of both factors thereafter throughout 21days. The seemingly low cumulative release of IL-10 was primarily due tothe spontaneous degradation of released IL-10 and molecules trapped inresidual coacervate. Coacervate delivery of FGF2 and IL-10 preservedtheir bioactivities on cardiac stromal cell proliferation in vitro.FGF2/IL-10 coacervate sustained HUVEC and hHP proliferation whilereducing CF proliferation in general, especially under the inflammatorystress condition.

Hearts treated with FGF2/IL-10 coacervate, Coa-F/I-500/500 inparticular, exhibited significantly improved long-term LV contractilefunction and ameliorated LV dilatation, suggesting the synergisticallytherapeutic efficacy by controlled delivery of FGF2 and IL-10.FGF2/IL-10 coacervate, especially Coa-F/I-500/500, augmented long-termrevascularization, particularly at the infarct area. The data providedin this Example also imply a positive correlation of revascularizingeffects with the dose of IL-10. In addition, coacervate containing FGF2and 500 ng IL-10 reduced LV fibrosis, preserved infarct wall thickness,and inhibited chronic phagocytic cell infiltration at the infarct area,more effective than coacervate loaded with FGF2 alone. These resultsfurther suggest the synergistic effects of coacervate with FGF2 andIL-10 in anti-fibrosis and anti-inflammation.

Moreover, Coa-F/I-500/100 and Coa-F/I-500/500 had substantiallyaugmented long-term LV myocardial elasticity, maintaining around 80% ofthe normal myocardial strain. Interestingly, Coa-F-500 treated heartsalso exhibited significantly increased myocardial elasticity. These datasuggest the primary effect of controlled, localized delivery of FGF2 onmyocardial elasticity, largely independent of IL-10 mediatedanti-inflammatory benefits. This is likely attributed to the enhancedfunctional revascularization and reduced cardiomyocyte death mediated bycontrolled release of FGF2-Putative mechanisms involved in FGF2/IL-10coacervate mediated ischemic heart repair are summarized in FIG. 12.

The estimation of the duration of coacervate treatment post-MI by MPEimaging indicates that injected coacervate had a temporal distributionof at least 4 weeks in situ. Consequently, FGF2, IL-10, or othertherapeutic proteins with high heparin affinity will likely persistwithin coacervate and increase their long-term bioavailability in thelocal tissue.

Overall, Coa-F/I-500/500 exhibited the highest therapeutic potentialamong all treatment groups. This warrants the pre-clinical translationof coacervate delivery of FGF2 in combination with IL-10 in large animalmodels. In addition, the application of FGF2/IL-10 coacervate for thetreatment of other ischemic conditions such as myocardial reperfusioninjury and peripheral artery disease demands future investigation.Currently we are investigating the dose-dependent effect(s) and theprecise mechanism(s) of anti-inflammation and immunomodulation mediatedby controlled release of IL-10.

In summary, heparin-based coacervate represents a promising vehicle forlocalized, controlled delivery of a combination of angiogenic andanti-inflammatory proteins. A single coacervate treatment with 500 ngeach of FGF2 and IL-10 resulted in long-term synergistic benefits in amouse MI model. Future study in pre-clinical large animal models iswarranted to evaluate its therapeutic potential for the treatingischemic heart disease. Given that heparin binds a wide range of trophicfactors, coacervate delivery of single or multiple therapeutic proteinscan be further expanded to applications in different pathologicalconditions.

Example 5—Treatment of Melanoma

Twenty four (24) C57BL/6 female mice were divided into three treatmentgroups: 1) protein coacervate; 3 doses; 2) Blank coacervate, and PBS(phosphate-buffered saline), n=4 for each group. Ten days prior to thefirst treatment, the mice were each inoculated with cells in saline attwo sites, one in each leg. Mice were randomized into groups and treatedwith 1st injection in only one leg (primary site). A second treatmentwas given seven (7) days subsequent to the first treatment. 50 μLinjection per mouse per treatment consists of 25 μL of IL-2 or IL-12,and 25 μL of 3.75 mg coacervate. IL-2 (interleukin-2, purchased fromPeproTech, Rocky Hill, N.J.) in 10 mM sodium citrate buffer with 0.1%BSA was dosed at 0.01 μg, 0.1 μg, and 1 μg per dose and mixed with 1.78μL of 150 mg/mL heparin in 0.9% saline and 23.22 μL of 150 mg/mL PEAD in0.9% saline. IL-12 (interleukin-12, purchased from PeproTech, RockyHill, N.J.) in 1.5×PBS with 0.1% BSA was dosed at 1 μg, 10 μg, and 30 μgper dose and mixed with heparin and PEAD in the same manner as IL-2coacervates. Tumor size was determined for each time point, and bloodwas collected for analysis (TBD).

As depicted in FIG. 13, IL-12 in mid- to high-doses prolong survival inmice. One mouse from the IL-12, 30 μg group died early (D4), which isbelieved to be due to the cancer overpowering the mouse before treatmenthad a chance to work. There is minimal difference across the IL-2 groupsand controls.

As shown in FIGS. 14 and 15A-15C, no significant difference was observedin tumor size across all groups, for both the primary and contralateralsites. It is noted that the drops in average tumor size were due to micedying, and therefore an artifact.

As shown in FIGS. 16 and 17A-17C, the highest dose (30 ug) yieldsminimal tumor growth for 3 out of 4 mice. As indicated above, one mousedied early on D4. The mid dose (10 ug) yields some effect but not aspronounced as the high dose; one mouse survived until D24 even thoughthe size of the tumor grew up to 576 mm². The low dose shows the leastdifference between the experimental and control groups.

While several examples and embodiments of the methods are describedhereinabove in detail, other examples and embodiments will be apparentto, and readily made by, those skilled in the art without departing fromthe scope and spirit of the invention. For example, it is to beunderstood that this disclosure contemplates that, to the extentpossible, one or more features of any embodiment can be combined withone or more features of any other embodiment. Accordingly, the foregoingdescription is intended to be illustrative rather than restrictive.

The following clauses illustrate various aspects of the invention.

Clause 1: A composition comprising a complex or coacervate of apolycationic polymer, a polyanionic polymer, and a cytokine selectedfrom an interferon and/or an interleukin.

Clause 2: The composition of clause 1, wherein the polyanionic is aheparin or heparan sulfate.

Clause 3: The composition of clause 1, wherein the polycationic polymeris a polymer composition comprising at least one moiety selected fromthe following:

-   -   (a)        [—OC(O)—CH(NHY)—CH₂—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]_(n),    -   (b)        [—OC(O)—CH₂—CH(NHY)—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]_(n),    -   (c)        [—OC(O)—CH(NHY)—CH₂—CH₂—C(O)O—CH2-CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]_(n),        and/or    -   (d)        [—OC(O)—CH₂—CH₂—CH(NHY)—C(O)O—CH2-CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂-]_(n),        wherein Y is —C(O)—CH(NH₃ ⁺)—(CH₂)₃—NH—C(NH₂)₂ ⁺ or —C(O)—CH(NH₃        ⁺)—(CH₂)₄—(NH₃)+, and R1 and R2 are the same or different and        are independently selected from the group consisting of        hydrogen, a carboxy-containing group, a C₁₋₆ alkyl group, an        amine-containing group, a quaternary ammonium containing group,        and a peptide.

Clause 4: The composition of clause 3, in which the polycationic polymeris a polymer composition comprising at least one moiety selected fromformulae (a) or (b).

Clause 5: The composition of clause 3, in which the polycationic polymeris a polymer composition comprising at least one moiety selected fromformulae (c) or (d).

Clause 6: The composition of any one of clauses 3-5, wherein thepolycationic polymer has a polydispersity index of less than 3.0.

Clause 7: The composition of any one of clauses 3-5, wherein thepolycationic polymer has a polydispersity index of less than 2.0.

Clause 8: The composition of any one of clauses 3-5, in which R1 and R2are selected from the group consisting of Ile-Lys-Val-Ala-Val (IKVAV)(SEQ ID NO: 1), Arg-Gly-Asp (RGD), Arg-Gly-Asp-Ser (RGDS) (SEQ ID NO:2), Ala-Gly-Asp (AGD), Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) (SEQ ID NO: 3),Val-Ala-Pro-Gly-Val-Gly (VAPGVG) (SEQ ID NO: 4), APGVGV (SEQ ID NO: 5),PGVGVA (SEQ ID NO: 6), VAP, GVGVA (SEQ ID NO: 7), VAPG (SEQ ID NO: 8),VGVAPG (SEQ ID NO: 9), VGVA (SEQ ID NO: 10), VAPGV (SEQ ID NO: 11) andGVAPGV (SEQ ID NO: 12).

Clause 9: The composition of any one of clauses 3-5, in which thepolycationic polymer is complexed with heparin or heparan sulfate.

Clause 10: The composition of any one of clauses 3-5, in which one orboth of R1 and R2 are maleate or phosphate.

Clause 11: The composition of any one of clauses 3-5, wherein Y is—C(O)—CH(NH₃ ⁺)—(CH₂)₄—(NH₃)⁺.

Clause 12: The composition of any one of clauses 3-5, wherein Y is—C(O)—CH(NH₃ ⁺)—(CH₂)₃—NH—C(NH₂)₂ ⁺.

Clause 13: The composition of any one of clauses 3-5, in which R1 ishydrogen.

Clause 14: The composition of any one of clauses 3-5, in which one orboth of R1 and R2 are charged.

Clause 15: The composition of any one of clauses 1-14, in which theratio of the polycationic polymer to the polyanionic polymer in thecomposition results in a neutral charge.

Clause 16: The composition of any one of clauses 1-14, in which theratio of the polycationic polymer to the polyanionic polymer in thecomposition results in a negative charge.

Clause 17: The composition of any one of clauses 1-14, in which theratio of the polycationic polymer to the polyanionic polymer in thecomposition results in a positive charge.

Clause 18: The composition of any one of clauses 1-17, wherein thecytokine is one or more cytokines selected from the group consisting ofan IL-2 (interleukin-2), an IL-12 (interleukin-12, e.g., IL-12 p70),and/or an IFN-γ (interferon gamma), in any combination.

Clause 19: The composition of any one of clauses 1-17, wherein thecytokine is an IL-12.

Clause 20: The composition of any one of clauses 1-17, wherein thecytokine is an immunomodulatory cytokine.

Clause 21: The composition of clause 20, wherein the cytokine is IL-10,and the composition further comprises an angiogenic growth factor.

Clause 22: The composition of clause 21, wherein the angiogenic growthfactor is FGF2.

Clause 23: The composition of any one of clauses 1-22, embedded in ahydrogel.

Clause 24: A method of delivering an interferon and/or an interleukin toa patient in need thereof, comprising administering the composition ofany of clauses 1-23 to the patient.

Clause 25: The method of clause 18, wherein the composition is deliveredby enteral, parenteral, or topical routes, for example and withoutlimitation by: intravenous (IV), local injection, intramuscular,intracerebral, subcutaneous, orally, inhalation, topically, enema,intravaginal, intrauterine, ocular, or otic routes.

Clause 26: A method of treating a cancer in a patient, comprising,delivering to the patient, e.g. by enteral, parenteral, or topicalroutes, for example and without limitation by: intravenous (IV), localinjection, intramuscular, intracerebral, subcutaneous, orally,inhalation, topically, enema, intravaginal, intrauterine, ocular, orotic routes, the composition of any of clauses 1-20.

Clause 27: The method of clause 26, wherein the cytokine is IL-12.

Clause 28: The method of clause 27 or 28, wherein the cancer ismelanoma.

Clause 29: A method of treating a myocardial infarct in a patient,comprising, delivering to the myocardium at or adjacent to the infarct acomposition of any one of clauses 20-23.

Clause 30: The method of clause 29, wherein the composition comprisesFGF2 and IL-10.

We claim:
 1. A composition comprising a complex or coacervate of apolycationic polymer, a polyanionic polymer, and one or more cytokinesand/or interferons selected from an IL-2, an IL-12, and/or an IFN-γ. 2.The composition of claim 1, wherein the polyanionic polymer is a heparinor heparan sulfate.
 3. The composition of claim 1, wherein thepolycationic polymer is a copolymer comprising a polyester backbonecomprising a copolymer of ethylene glycol diglyceride and eitheraspartic acid or glutamic acid, and pendant arginine groups.
 4. Thecomposition of claim 3, wherein the polycationic polymer has apolydispersity index of less than 3.0.
 5. The composition of claim 1, inwhich the ratio of the polycationic polymer to the polyanionic polymerin the composition results in a neutral charge.
 6. The composition ofclaim 1, in which the ratio of the polycationic polymer to thepolyanionic polymer in the composition results in a negative charge. 7.The composition of claim 1, in which the ratio of the polycationicpolymer to the polyanionic polymer in the composition results in apositive charge.
 8. The composition of claim 1, wherein the polycationicpolymer is a copolymer comprising a polyester backbone comprising acopolymer of ethylene glycol diglyceride and either aspartic acid orglutamic acid, and pendant lysine groups.
 9. The composition of claim 1,wherein the polycationic polymer is poly(ethylene arginylaspartatediglyceride).
 10. The composition of claim 1, wherein the polycationicpolymer is poly(ethylene lysinylaspartate diglyceride).
 11. Thecomposition of claim 1, wherein the cytokine is IL-2.
 12. Thecomposition of claim 1, wherein the cytokine is IL-12.
 13. Thecomposition of claim 1, wherein the interferon is IFN-γ.
 14. Thecomposition of claim 1, embedded in a hydrogel.
 15. A method ofdelivering an interleukin to a patient in need thereof, comprisingadministering the composition of claim 1 to the patient.
 16. A method oftreating a cancer in a patient, comprising, delivering to the patientthe composition of claim
 1. 17. The method of claim 16, wherein thecancer is melanoma.